Methods and compositions for identifying downy mildew resistant cucumber plants

ABSTRACT

The present invention relates to methods for identifying cucumber lines having increased resistance to Downy Mildew, and identification of genetic markers linked to gene(s) conditioning such increased disease resistance. The present invention also relates to methods of breeding cucumber plants from lines having increased Downy Mildew resistance by marker-assisted selection, compositions including nucleic acid probes or primers which are useful for such marker assisted selection, and plants and plant parts produced by such methods.

BACKGROUND OF THE INVENTION

This application claims the priority of U.S. Provisional Appl. Ser. No.61/254,141, filed Oct. 22, 2009, the entire disclosure of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMS108US_ST25.txt”, which is 30,174 bytes (measured in MS-WINDOWS),created on Oct. 21, 2010 is filed herewith by electronic submission andis incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods and compositions for identifying andbreeding of cucumber plants having Downy Mildew resistance.

BACKGROUND OF THE INVENTION

Cucumber (Cucumis sativus L.) is a popular vegetable crop that has beencultivated for several thousand years and is grown worldwide. Cucumberplants are grown in a wide range of climates, and in open fields as wellas greenhouses. The two main types of cucumber fruit grown commerciallytoday are fresh market (slicing) and processing (pickling).

Downy Mildew (DM) is caused by the fungus Pseudoperonospora cubensis(P.c.), which causes significant crop losses among many Cucurbitspecies, including cucumber. The disease is found worldwide and favorsmoist, temperate conditions. The disease affects greenhouse grownplants, and plants grown in the field. DM is one of the most importantfoliar diseases of cucurbits, and can reduce fruit yield and quality,and may kill susceptible seedlings.

Symptoms of DM infection are variable. Initial symptoms include sharp,irregular yellow lesions on the upper surface of the leaves, whicheventually become more distinct on both sides of the leaves. Theunderside of the leaves may exhibit a whitish-gray, brown, or light bluegrowth, particularly under moist conditions. This downy growth is sporesproduced on the lower surface of the lesion. A general yellowing ofaffected leaves typically occurs as the lesions coalesce into one largelesion, eventually causing the leaf to wilt and die. The disease canprogress quite rapidly, killing foliage in a matter of a few days andresulting in poor fruit production and quality. Cucumber fruit are notaffected directly, but major defoliation exposes the fruit to sunscald.Once it appears on a crop, DM rapidly spreads by wind, or splashing rainand/or irrigation water. Disease management and prevention requiresdestruction of all plants from infected nurseries and disinfection ofthe facilities. Emergence of a new isolate of DM has also overcome somepreviously known resistant lines. Thus, there is a need for new cucumbervarieties having resistance to DM, and methods for producing suchplants.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method of obtaining cucumbergermplasm comprising the steps of: a) assaying cucumber plants for thepresence of at least a first genetic marker genetically linked to a QTLthat confers resistance to Downy Mildew; and b) selecting at least afirst cucumber plant comprising the genetic marker and the QTL thatconfers resistance to Downy Mildew; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0228457 and SNP markerNN0228148, which map to approximately 25.2 cM and 82.7 cM on the geneticmap of the linkage group termed cucumber chromosome 5; wherein the QTLmaps to a position between the sequence represented by SNP markerNN0225012 and SNP marker NN0227587 which map to approximately 20.6 cMand 87.4 cM on the genetic map of the linkage group termed cucumberchromosome 4; or wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0223782 and SNP marker NN0226638 which mapto approximately 22.5 cM and 88.4 cM on the genetic map of the linkagegroup termed cucumber chromosome 2. In particular embodiments, the QTLallele which confers resistance to Downy Mildew is derived from cucumberline PI197088, or a progeny plant thereof.

In certain embodiments, the QTL maps to a position between the sequencerepresented by SNP marker NN0227981 and SNP marker NN0226631, which mapto approximately 35.7 cM and 55.5 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0227981 andSNP marker NN0247786, which map to approximately 35.7 cM and 71.4 cM onthe genetic map of the linkage group termed cucumber chromosome 5. Inother embodiments, the QTL maps to a position between the sequencerepresented by SNP marker NN0228579 and SNP marker NN0224495 which mapto approximately 25.8 cM and 82.0 cM on the genetic map of the linkagegroup termed cucumber chromosome 4; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0225088 and SNP markerNN0224041 which map to approximately 30.4 cM and 75.8 cM on the geneticmap of the linkage group termed cucumber chromosome 4; or wherein theQTL maps to a position between the sequence represented by SNP markerNN0228579 and SNP marker NN0224495 which map to approximately 54.5 cMand 82.0 cM on the genetic map of the linkage group termed cucumberchromosome 4. In yet another embodiment, the QTL maps to a positionbetween the sequence represented by SNP marker NN0246378 and SNP markerNN0246472 which map to approximately 14.6 cM and 38.4 cM on the geneticmap of the linkage group termed cucumber chromosome 2.

In certain embodiments, the invention also provides such a method,wherein selecting the first cucumber plant further comprises selectingthe plant based on the presence of a plurality of genetic markers thatmap to a position between the sequence represented by SNP markerNN0228457 and SNP marker NN0228148, which map to approximately 25.2 cMand 82.7 cM on the genetic map of the linkage group termed cucumberchromosome 5; or wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0227981 and SNP marker NN0226631, which mapto approximately 35.7 cM and 55.5 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0227981 andSNP marker NN0247786, which map to approximately 35.7 cM and 71.4 cM onthe genetic map of the linkage group termed cucumber chromosome 5; orwherein the QTL maps to a position between the sequence represented bySNP marker NN0225012 and SNP marker NN0227587 which map to approximately20.6 cM and 87.4 cM on the genetic map of the linkage group termedcucumber chromosome 4; or wherein the QTL maps to a position between thesequence represented by SNP marker NN0247342 and SNP marker NN0224495which map to approximately 54.5 cM and 82.0 cM on the genetic map of thelinkage group termed cucumber chromosome 4; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0223782 andSNP marker NN0226638 which map to approximately 22.5 cM and 88.4 cM onthe genetic map of the linkage group termed cucumber chromosome 2.

In certain embodiments, the genetic marker is selected from the groupconsisting of markers NN0223782, NN0225385, NN0226670, NN0224124,NN0246472, NN0225358, NN0227700, NN0224617, NN0247695, NN0227242,NN0223824, NN0223181, NN0226638, NN0225012, NN0228579, NN0226451,NN0225088, NN0226219, NN0247551, NN0246357, NN0225551, NN0226732,NN0247689, NN0247342, NN0224702, NN0225482, NN0224538, NN0247543,NN0224041, NN0228853, NN0227762, NN0227587, NN0228457, NN0246356,NN0246332, NN0223399, NN0223689, NN0247731, NN0226166, NN0225480,NN0246411, NN0227759, NN0247348, NN0228465, NN0247786, NN0226645,NN0223809, NN0227071, NN0226870, NN0228148, NN0246378, NN0224041,NN0227587, NN5096749, NN0224856, NN0223160, NN0223809, NN0226631,NN0227981, NN0247786, NN0246425, NN0224495, NN0225088, NN0227071,NN0223782, NN0228579, NN0247342, and NN0247731, comprising a singlenucleotide polymorphism of one of SEQ ID NOs:20-87. In particularembodiments the genetic marker is selected from the group consisting ofmarkers NN0223782, NN0225385, NN0226670, NN0224124, NN0246472,NN0225358, NN0227700, NN0224617, NN0247695, NN0227242, NN0223824,NN0223181, NN0226638, and NN0246378. In yet other embodiments, thegenetic marker is selected from the group consisting of markersNN0225012, NN0228579, NN0226451, NN0225088, NN0226219, NN0247551,NN0246357, NN0225551, NN0226732, NN0247689, NN0247342, NN0224702,NN0225482, NN0224538, NN0247543, NN0224041, NN0228853, NN0227762,NN0227587, NN0224041, NN0227587, NN0246425, NN0224495, NN0225088,NN0228579, and NN0247342. In still yet other embodiments, the geneticmarker is selected from the group consisting of markers NN0228457,NN0246356, NN0246332, NN0223399, NN0223689, NN0247731, NN0226166,NN0225480, NN0246411, NN0227759, NN0247348, NN0228465, NN0247786,NN0226645, NN0223809, NN0227071, NN0226870, NN0228148, NN5096749,NN0224856, NN0223160, NN0223809, NN0226631, NN0227981, NN0247786,NN0227071, and NN0247731. In particular embodiments, the genetic markeris NN0226631 or NN0246425. Further, in such embodiments assaying thecucumber plants comprises PCR, single strand conformational polymorphismanalysis, denaturing gradient gel electrophoresis, cleavage fragmentlength polymorphism analysis, TAQMAN assay, and/or DNA sequencing.

Further, in certain embodiments the genetic marker may map within 20 cM,10 cM or 1 cM of a QTL which confers resistance to Downy Mildew

In another aspect, the invention provides a method of cucumber plantbreeding comprising: a) assaying cucumber plants for the presence of atleast a first genetic marker genetically linked to a QTL that confersresistance to Downy Mildew; and b) selecting at least a first cucumberplant comprising the genetic marker and the QTL that confers resistanceto Downy Mildew; wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0228457 and SNP marker NN0228148, which mapto approximately 25.2 cM and 82.7 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0227981 and SNP markerNN0226631, which map to approximately 35.7 cM and 55.5 cM on the geneticmap of the linkage group termed cucumber chromosome 5; wherein the QTLmaps to a position between the sequence represented by SNP markerNN0227981 and SNP marker NN0247786, which map to approximately 35.7 cMand 71.4 cM on the genetic map of the linkage group termed cucumberchromosome 5; wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0225012 and SNP marker NN0227587 which mapto approximately 20.6 cM and 87.4 cM on the genetic map of the linkagegroup termed cucumber chromosome 4; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0247342 and SNP markerNN0224495 which map to approximately 54.5 cM and 82.0 cM on the geneticmap of the linkage group termed cucumber chromosome 4; or wherein theQTL maps to a position between the sequence represented by SNP markerNN0223782 and SNP marker NN0226638 which map to approximately 22.5 cMand 88.4 cM on the genetic map of the linkage group termed cucumberchromosome 2; and c) crossing the first cucumber plant with itself or asecond cucumber plant to produce progeny cucumber plants comprising theQTL that confers resistance to Downy Mildew.

In one embodiment of the method, selecting at least a first cucumberplant further comprises selecting the plant based on the presence of aplurality of genetic markers that map to a position between the sequencerepresented by SNP marker NN0228457 and SNP marker NN0228148, which mapto approximately 25.2 cM and 82.7 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0227981 and SNP markerNN0226631, which map to approximately 35.7 cM and 55.5 cM on the geneticmap of the linkage group termed cucumber chromosome 5; wherein the QTLmaps to a position between the sequence represented by SNP markerNN0227981 and SNP marker NN0247786, which map to approximately 35.7 cMand 71.4 cM on the genetic map of the linkage group termed cucumberchromosome 5; wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0225012 and SNP marker NN0227587 which mapto approximately 20.6 cM and 87.4 cM on the genetic map of the linkagegroup termed cucumber chromosome 4; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0247342 and SNP markerNN0224495 which map to approximately 54.5 cM and 82.0 cM on the geneticmap of the linkage group termed cucumber chromosome 4; or wherein theQTL maps to a position between the sequence represented by SNP markerNN0223782 and SNP marker NN0226638 which map to approximately 22.5 cMand 88.4 cM on the genetic map of the linkage group termed cucumberchromosome 2. In another embodiment of the method, it may furthercomprise the step of: d) selecting a progeny plant comprising the allelewhich confers resistance to Downy Mildew and crossing the progeny plantwith itself or a third cucumber plant to produce additional progenyplants. In certain embodiments, the method further comprises repeatingstep (d) about 2-10 times. Likewise, in certain embodiments the allelewhich confers resistance to Downy Mildew is derived from cucumber linePI197088, or a progeny plant thereof.

In certain embodiments, the genetic marker maps within 20 cM, 10 cM or 1cM of a QTL which confers resistance to Downy Mildew. In someembodiments the genetic marker is selected from the group consisting ofmarkers NN0223782, NN0225385, NN0226670, NN0224124, NN0246472,NN0225358, NN0227700, NN0224617, NN0247695, NN0227242, NN0223824,NN0223181, NN0226638, NN0225012, NN0228579, NN0226451, NN0225088,NN0226219, NN0247551, NN0246357, NN0225551, NN0226732, NN0247689,NN0247342, NN0224702, NN0225482, NN0224538, NN0247543, NN0224041,NN0228853, NN0227762, NN0227587, NN0228457, NN0246356, NN0246332,NN0223399, NN0223689, NN0247731, NN0226166, NN0225480, NN0246411,NN0227759, NN0247348, NN0228465, NN0247786, NN0226645, NN0223809,NN0227071, NN0226870, NN0228148, NN0246378, NN0224041, NN0227587,NN5096749, NN0224856, NN0223160, NN0223809, NN0226631, NN0227981,NN0247786, NN0246425, NN0224495, NN0225088, NN0227071, NN0223782,NN0228579, NN0247342, and NN0247731, comprising a single nucleotidepolymorphism of one of SEQ ID NOs:20-87. In particular embodiments ofthe method, the genetic marker is selected from the group consisting ofmarkers NN0223782, NN0225385, NN0226670, NN0224124, NN0246472,NN0225358, NN0227700, NN0224617, NN0247695, NN0227242, NN0223824,NN0223181, NN0226638, and NN0246378. In other embodiments the geneticmarker is selected from the group consisting of markers NN0225012,NN0228579, NN0226451, NN0225088, NN0226219, NN0247551, NN0246357,NN0225551, NN0226732, NN0247689, NN0247342, NN0224702, NN0225482,NN0224538, NN0247543, NN0224041, NN0228853, NN0227762, NN0227587,NN0224041, NN0227587, NN0246425, NN0224495, NN0225088, NN0228579, andNN0247342. In yet other embodiments, the genetic marker is selected fromthe group consisting of markers NN0228457, NN0246356, NN0246332,NN0223399, NN0223689, NN0247731, NN0226166, NN0225480, NN0246411,NN0227759, NN0247348, NN0228465, NN0247786, NN0226645, NN0223809,NN0227071, NN0226870, NN0228148, NN5096749, NN0224856, NN0223160,NN0223809, NN0226631, NN0227981, NN0247786, NN0227071, and NN0247731. Inparticular embodiments, the genetic marker is NN0226631 or NN0246425. Insuch embodiments, assaying the cucumber plants may comprise PCR, singlestrand conformational polymorphism analysis, denaturing gradient gelelectrophoresis, cleavage fragment length polymorphism analysis, TAQMANassay, and/or DNA sequencing.

In some embodiments the cucumber plant comprising at least one allelewhich confers resistance to Downy Mildew demonstrates a reduction offoliar symptoms of chlorotic and/or necrotic lesions of at least, orgreater than, 25%, relative to a non-resistant control cucumber line.

In another aspect, the invention provides an isolated nucleic acid probeor primer that hybridizes under conditions of 5×SSC, 50% formamide, and42° C. to a cucumber plant genomic region mapping within 40 cM of a QTLwhich confers resistance to Downy Mildew and comprises a sequence whichmaps on cucumber chromosomes 2, 4, or 5, wherein the probe or primercomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:20-87.

In another aspect, a cucumber plant is provided which is produced by themethod of: a) assaying cucumber plants for the presence of at least afirst genetic marker genetically linked to a QTL that confers resistanceto Downy Mildew; and b) selecting at least a first cucumber plantcomprising the genetic marker and the QTL that confers resistance toDowny Mildew; wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0228457 and SNP marker NN0228148, which mapto approximately 25.2 cM and 82.7 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0227981 and SNP markerNN0226631, which map to approximately 35.7 cM and 55.5 cM on the geneticmap of the linkage group termed cucumber chromosome 5; wherein the QTLmaps to a position between the sequence represented by SNP markerNN0227981 and SNP marker NN0247786, which map to approximately 35.7 cMand 71.4 cM on the genetic map of the linkage group termed cucumberchromosome 5; wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0225012 and SNP marker NN0227587 which mapto approximately 20.6 cM and 87.4 cM on the genetic map of the linkagegroup termed cucumber chromosome 4; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0247342 and SNP markerNN0224495 which map to approximately 54.5 cM and 82.0 cM on the geneticmap of the linkage group termed cucumber chromosome 4; or wherein theQTL maps to a position between the sequence represented by SNP markerNN0223782 and SNP marker NN0226638 which map to approximately 22.5 cMand 88.4 cM on the genetic map of the linkage group termed cucumberchromosome 2; and c) crossing the first cucumber plant with itself or asecond cucumber plant to produce progeny cucumber plants comprising theQTL that confers resistance to Downy Mildew. Further provided is aprogeny plant thereof that comprises said genetic marker and QTL thatconfers resistance to Downy Mildew. The invention also provides, infurther embodiments, such a cucumber plant comprising two introgressedcucumber chromosomal regions conferring resistance to Pseudoperonosporacubensis, wherein the regions comprise a Downy Mildew resistancecontributing QTL region found on chromosome 4, and a Downy Mildewresistance contributing QTL region found on chromosome 5. In particularembodiments the cucumber plant is homozygous for said chromosomal regionor regions.

In certain embodiments the cucumber plant, or progeny plant thereof, isfurther defined as an agronomically elite plant. Also provided incertain embodiments is a part of such a cucumber plant or progenythereof, further defined as pollen, an ovule, a leaf, an embryo, a root,a root tip, an anther, a flower, a fruit, a stem, a shoot, a seed, aprotoplast, a cell, and a callus. Another embodiment provides a seedthat produced such a plant.

Certain embodiments provide a cucumber plant comprising at least a firstintrogressed cucumber chromosomal region conferring resistance toPseudoperonospora cubensis, wherein the region is selected from thegroup consisting of: a Downy Mildew resistance contributing QTL regionfound on chromosome 2, a Downy Mildew resistance contributing QTL regionfound on chromosome 4, and a Downy Mildew resistance contributing QTLregion found on chromosome 5; further wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0228457 and SNP markerNN0228148, which map to approximately 25.2 cM and 82.7 cM on the geneticmap of the linkage group termed cucumber chromosome 5; wherein the QTLmaps to a position between the sequence represented by SNP markerNN0225012 and SNP marker NN0227587 which map to approximately 20.6 cMand 87.4 cM on the genetic map of the linkage group termed cucumberchromosome 4; or wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0246378 and SNP marker NN0247695 which mapto approximately 14.6 cM and 66.5 cM on the genetic map of the linkagegroup termed cucumber chromosome 2. Particular embodiments provide acucumber plant, wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0227981 and SNP marker NN0226631, which mapto approximately 35.7 cM and 55.5 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0227981 andSNP marker NN0247786, which map to approximately 35.7 cM and 71.4 cM onthe genetic map of the linkage group termed cucumber chromosome 5. Infurther embodiments, the invention provides a cucumber plant comprisingat least two introgressed cucumber chromosomal regions selected fromsaid group.

In some embodiments the QTL of the cucumber plant maps to a positionbetween the sequence represented by SNP marker NN0228579 and SNP markerNN0224495 which map to approximately 25.8 cM and 82.0 cM on the geneticmap of the linkage group termed cucumber chromosome 4; wherein the QTLmaps to a position between the sequence represented by SNP markerNN0225088 and SNP marker NN0224041 which map to approximately 30.4 cMand 75.8 cM on the genetic map of the linkage group termed cucumberchromosome 4; or wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0228579 and SNP marker NN0224495 which mapto approximately 54.5 cM and 82.0 cM on the genetic map of the linkagegroup termed cucumber chromosome 4. In other embodiments the QTL maps toa position between the sequence represented by SNP marker NN0246378 andSNP marker NN0246472 which map to approximately 14.6 cM and 38.4 cM onthe genetic map of the linkage group termed cucumber chromosome 2.

Also provided is a cucumber plant wherein the first introgressedcucumber chromosomal region conferring resistance to Pseudoperonosporacubensis comprises an allele present in PI197088. In certainembodiments, the cucumber plant comprises a QTL region found onchromosome 2, wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0246378 and SNP marker NN0247695 which mapto approximately 14.6 cM and 66.5 cM on the genetic map of the linkagegroup termed cucumber chromosome 2, and wherein the QTL is introgressedfrom PI197088. The cucumber plant may further be defined as comprisingan allele from PI197088 at one or more of markers NN0246378, NN0223782,NN0246472, and NN0247695. In particular embodiments the cucumber plantis further defined as comprising at least a first allele not present inPI197088, wherein the allele is detected with the group of markerscomprising NN0247695, NN0246472, NN0223782, and NN0246378.

In other embodiments of the invention, the cucumber plant comprises theDowny Mildew resistance contributing QTL region found on chromosome 4wherein the QTL maps to a position between the sequence represented bySNP marker NN0225012 and SNP marker NN0227587 which map to approximately20.6 cM and 87.4 cM on the genetic map of the linkage group termedcucumber chromosome 4. In certain embodiments the DM resistance QTL isintrogressed from PI197088. In particular embodiments the cucumber plantmay further be defined as comprising: a) an allele from PI197088 at oneor more of markers selected from the group consisting of: NN0228579,NN0225088, NN0225551, NN0247342, NN0246425, NN0224041, NN0224495, andNN0227587; b) an allele which is not present in PI197088 of at least onemarker selected from the group consisting of: NN0228579, NN0225088,NN0225551, NN0247342, NN0246425, NN0224041, NN0224495, and NN0227587; c)an allele from PI197088 at marker NN0246425, an allele not present inPI197088 at marker NN0247342 and an allele not present in PI197088 atmarker NN0224495; d) an allele from PI197088 at marker NN0246425, anallele not present in PI197088 at marker NN0225088 and an allele notpresent in PI197088 at marker NN0224495; or e) an allele from PI197088at marker NN0246425, an allele not present in PI197088 at markerNN0228579, and an allele not present in PI197088 at marker NN0224495.

In other embodiments, the cucumber plant of claim 36, comprising the QTLregion found on chromosome 5, wherein the QTL maps to a position betweenthe sequence represented by SNP marker NN0228457 and SNP markerNN0228148, which map to approximately 25.2 cM and 82.7 cM on the geneticmap of the linkage group termed cucumber chromosome 5. In certainembodiments the QTL region is introgressed from PI197088. In particulaembodiments the cucumber plant may further be defined as comprising: a)an allele from PI197088 at one or more of markers selected from thegroup consisting of: NN5096749, NN0224856, NN0227981, NN0247731,NN0226631, NN0247786, NN0223160, NN0223809, NN0227071, and NN0228148; b)an allele which is not present in PI197088 of at least one markerselected from the group consisting of: NN0228148, NN0227071, NN0223809,NN0223160, NN0247786, NN0226631, NN0247731, NN0227981, NN0224856, andNN5096749; c) an allele from PI197088 at marker NN0226631, an allele notpresent in PI197088 at marker NN0227981, and an allele not present inPI197088 at marker NN0247786; d) an allele from PI197088 at markerNN0226631, an allele not present in PI197088 at marker NN0227981, and anallele not present in PI197088 at marker NN0227071; or e) an allele fromPI197088 at marker NN0226631, an allele not present in PI197088 atmarker NN0224856, and an allele not present in PI197088 at markerNN0227071.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a genetic map (left) and LOD plot (right) for markereffects on Downy Mildew reaction in 148 F3 families from the cucumberL088 (Lucinde×PI197088) population. Lines between the genetic map andLOD plot provide reference positions for three of the markers to aid incomparisons of map position and LOD score. This linkage groupcorresponds to chromosome 5 as described for instance in Ren et al.,2009 (PLoS ONE 4:e5795, 2009; doi:10.1371/journal.pone.0005795). Otherexemplary genetic maps of cucumber are provided for instance in Staub,et al., 2007 (HortScience, 40:20-27), and Meboah et al., 2007 (Afr. J.Biotechnol. 6:2784-2791).

FIG. 2 depicts Downy Mildew resistance data for certain additionalmarkers in an identified Downy Mildew resistance contributing QTLregion, showing correlation between DM resistance and marker position.

FIG. 3 depicts typical disease reactions for (A) PI 197088; (B) cv.Adefem; and (C) cv. Maram.

FIG. 4 depicts typical disease reactions on mature leaves whichrepresent the disease rating scale outlined in Example 6, where “1” ismost resistant and “9” is most susceptible.

FIG. 5 depicts genetic maps of cucumber chromosomes 5, 4, and 2 withnumerous SNP markers (“NNO . . . ”) as well as previously utilized RAPDor CAPS markers such as CAPS_ENK59. The number on the left of eachschematic chromosome represents genetic distance in cM from the “top” ofthe map. The marker locations are given on the right of each diagram.The thick two-sided arrows to the right of the designated markersrepresent the approximate location of QTL 1, 2, and 4. (A) Chromosome 5from the API mapping population; (B) Chromosome 5 from the VJ mappingpopulation; (C) Chromosome 4 from the API mapping population; (D)Chromosome 4 from the VJ mapping population; (E) Chromosome 2 from theAPI mapping population; (F) Chromosome 2 from the VJ mapping population.

FIG. 6 Interval mapping and LOD scores for QTLs of chromosomes 2, 4, and5, analyzed in F4 and F5 generations of the QIR mapping population.Shown are the LOD scores from the single-QTL mapping analysis forphenotypes collected from the F₄ (solid line) and F₅ (dotted line)generations of the QIR mapping population. The two horizontal linesindicate the α=0.05 permutation thresholds from 1,000 permutations forthe two datasets. “Chr” referens to chromosome.

FIG. 7. Plots of F₄ and F₅ Downy Mildew pathogenicity test scores in theQIR mapping population, means against genotypes at the correspondingQTLs. The QTL identified from the F₄ data was at Chr5:55.5 cM and theQTL identified from the F₅ data was at Chr4:69.8 cM. Error barscorrespond to ±1 standard error.

FIG. 8. Interaction plots between the two QTLs for Downy mildewresistance in the QIR population. In each plot, the three genotypes onthe x-axis correspond to the QTL identified using the correspondingdatasets. The three genotypes (AA, AB, and BB) of the alternate QTL areindicated by solid, dashed, or dotted lines, respectively. Error barscorrespond to ±1 standard error.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for identifying cucumber plants (Cucumissativus) having resistance to Downy Mildew (DM) caused byPseudoperonospora cubensis. Such cucumber lines can be referred to as DMresistant cucumber varieties. Methods of breeding DM resistant cucumberlines are further provided. Also disclosed herein are molecular markersthat are linked to quantitative trait loci contributing to DMresistance. Through use of the markers, one of skill in the art mayincrease the degree of DM resistance in cucumber or select plants for anincreased predisposition for DM resistance. In particular embodiments,the methods are performed on progeny cucumber plants of cucumber linePI197088, such as members of the API, VJ, or QIR mapping populationsdisclosed herein, or progeny thereof. The QTLs identified in this mannermay be combined with one or more other QTLs that also contribute to DMresistance, as desired. In addition to the QTL identified in FIG. 1which corresponds to “QTL 1” (localized to chromosome 5:25.3-82.1 cM asidentified in FIGS. 5A-5B and Table 19; or chromosome 5:25.2-78.3 cM, orchromosome 5:28.5-75.0 cM), another major QTL (termed “QTL 2”) wasidentified in the API, VJ, or QIR mapping populations, at chromosome4::20.6-87.4 cM (see FIGS. 5C-5D and Table 19; or chromosome 4:25.8-82.0cM; or chromosome 4:31.8-75.8 cM), also having significant effect onDowny Mildew resistance in cucumber. Individually, each allele of thesetwo QTLs is estimated to have the potential to significantly reduce aplant's Downy Mildew disease rating in a DM pathology test as describedherein, for instance in both the API and VJ mapping population geneticbackgrounds, when grown in multiple tested geographic locations. Anadditional QTL (“QTL 4”; see FIGS. 5E-5F) mapping to chromosome 2:22.3-88.4 cM also has an effect in cucumber Downy Mildew resistance,although its genetic effect is apparently more complex, with potentialinteractions with other QTLs and/or the growth environment.

The average individual additive allelic effects are estimated at 0.62(0.32-1.26) at QTL 1 and 0.62 (0.47-0.93) at QTL 2, from the studiesdescribed in Example 8. Allelic effects of about 0.7 disease score unitsat each of QTL1 and QTL2 of chromosomes 4 and 5 were observed in the QIRmapping population. Therefore, an individual plant with both resistantalleles at both QTLs is likely to have a reduction in disease rating of0.62×2+0.62×2=2.48. Individually, the average amount of phenotypicvariation explained by each of these two QTLs is 24% (QTL 1) and 21%(QTL 2), with the remaining 76%-79% attributable to other geneticeffects and environmental (non-genetic) effects.

The definition of these QTLs allows the use of specific molecularmarkers, such as those disclosed herein, in a plant breeding program tointrogress a Downy Mildew Resistance trait or traits to agronomicallyacceptable cucumber lines. Marker-assisted introgression involves thetransfer of a chromosomal region, defined by one or more markers, fromone germplasm to a second germplasm. An initial step in that process isthe localization of the trait by gene mapping which is the process ofdetermining the position of a gene relative to other genes and geneticmarkers through linkage analysis. The basic principle for linkagemapping is that the closer together two genes are on the chromosome, themore likely they are to be inherited together. Briefly, a cross is madebetween two genetically compatible but divergent parents relative to atrait under study (e.g. DM resistance). Genetic markers are then used tofollow the segregation of traits under study in the progeny from thecross, often termed a “mapping population.” The current inventionrelates to the introgression in cucumber of genetic material, e.g.,mapping to one or more QTLs, which is capable of causing a plant to bemore resistant to the pathogen which causes cucumber Downy mildewdisease. The present inventors have identified chromosomal regionsresponsible for enhanced DM resistance and used marker assisted breedingto introgress these specific linkage blocks into other cucumbergermplasm which lacked such resistance to DM. In certain embodiments ofthe invention, the process for producing DM resistant cucumber plant orline comprises introgressing at least one chromosomal locus mapping toQTL 1, QTL 2, and/or QTL 4 from a more DM resistant cucumber plant,line, or variety into a less DM resistant cucumber plant, line, orvariety. In specific embodiments, the more DM resistant cucumber plant,line, or variety is PI197088, or a progeny plant thereof.

Introgression of a particular DNA element or set of elements into aplant genotype is defined as the result of the process of backcrossconversion. A plant genotype into which a DNA sequence has beenintrogressed may be referred to as a backcross converted genotype, line,or variety. Such genotype, line, or variety may be an inbred or a hybridgenotype, line, or variety. Similarly a plant genotype lacking saiddesired DNA sequence may be referred to as an unconverted genotype,line, or variety. During breeding, the genetic markers linked toenhanced DM resistance may be used to assist in breeding for the purposeof producing cucumber plants with increased resistance toPseudoperonospora cubensis. A skilled worker would understand that theintrogression of a DM resistance trait into a cucumber plant may bemonitored by visual clues such as by use of a disease resistance testwith a disease rating scale as described herein, and/or by monitoringand breeding for the presence of molecular markers as described herein(i.e. marker assisted selection).

Localization of such markers to specific genomic regions or contigsfurther allows for use of associated sequences in breeding, to developadditional linked genetic markers, as well as to identify the mechanismfor resistance at more precise genetic and biochemical levels. It willbe understood to those of skill in the art that other markers or probeswhich more closely map the chromosomal regions as identified hereincould be employed to identify plants comprising a desired QTL for DMresistance. The chromosomal regions of the present invention facilitateintrogression of increased DM resistance from DM resistant germplasm,such as PI197088 or progeny thereof, into other germplasm, preferablyagronomically useful cucumber germplasm. Linkage blocks of various sizescould be transferred within the scope of this invention as long as thechromosomal region enhances the DM resistance of a desirable cucumberplant, line, or variety. Accordingly, it is emphasized that the presentinvention may be practiced using any molecular markers which geneticallymap in similar regions, provided that the markers are polymorphicbetween the parents.

In particular embodiments, these markers may be genetically linked tothe described QTLs for DM resistance which are located on cucumberchromosomes 2, 4, or 5, for instance as defined in the genetic map ofRen et al. (PLoS ONE 4:e5795, 2009; doi:10.1371/journal.pone.0005795).In certain embodiments, the markers are within 50 cM, 45 cM, 40 cM, 30cM, 20 cM, 10 cM, 5 cM, 3 cM, 1 cM, or less, of QTL 1 defined onchromosome 5, at 22.6-81.0 cM; or QTL 2 defined on chromosome 4 at37.7-87.6 cM; or QTL 4 defined on chromosome 2 at 21.8-73.8 cM, based onanalysis of the API and VJ mapping populations as described herein. Inparticular embodiments, the markers used to follow the presence of anyof these QTLs for DM resistance which are located on cucumberchromosomes 5, 4, or 2, are selected from the group consisting of:NN0223782, NN0225385, NN0226670, NN0224124, NN0246472, NN0225358,NN0227700, NN0224617, NN0247695, NN0227242, NN0223824, NN0223181,NN0226638, NN0225012, NN0228579, NN0226451, NN0225088, NN0226219,NN0247551, NN0246357, NN0225551, NN0226732, NN0247689, NN0247342,NN0224702, NN0225482, NN0224538, NN0247543, NN0224041, NN0228853,NN0227762, NN0227587, NN0228457, NN0246356, NN0246332, NN0223399,NN0223689, NN0247731, NN0226166, NN0225480, NN0246411, NN0227759,NN0247348, NN0228465, NN0247786, NN0226645, NN0223809, NN0227071,NN0226870, NN0228148, NN0246378, NN0224041, NN0227587, NN5096749,NN0224856, NN0223160, NN0223809, NN0226631, NN0227981, NN0247786,NN0246425, NN0224495, NN0225088, NN0227071, NN0223782, NN0228579,NN0247342, and NN0247731, comprising a single nucleotide polymorphism ofone of SEQ ID NOs:20-87 as shown in Tables 13 and 22, and UBC12-1200,and CAPs_ENK59, or other genetic markers linked to any of these QTLs.The presence of alleles conferring resistance to DM may be identified byuse of well known techniques, such as by nucleic acid detection methodsutilizing probes or primers comprising a sequence selected from thegroup consisting of SEQ ID NO:1-87. In certain embodiments, the methodcomprises detecting the presence of one or more single nucleotidepolymorphisms (SNP's) given in one or more of SEQ ID NOs:20-87.

In certain embodiments, the DM resistance QTL of chromosome 2 is definedas spanning the region defined by SNP marker NN0223782 (map position22.5 according to Table 19), to SNP marker NN0226638 (map position88.4). In other embodiments, the DM resistance QTL of chromosome 2 isdefined as spanning the region defined by SNP marker NN0246378 (mapposition ˜14.6 according to Tables 19 or 22), to SNP marker NN0246472(map position 38.4 according to Table 19).

In certain embodiments, the DM resistance QTL of chromosome 4 is definedas spanning the region defined by SNP marker NN0225012 (map position20.6), to SNP marker NN0227587 (map position 87.4). In otherembodiments, the DM resistance QTL of chromosme 4 is defined as spanningthe region defined by SNP marker NN225088 (map position 30.4) to SNPmarker NN0224041 (map position 75.8). In other embodiments, the DMresistance QTL of chromosome 4 is defined as spanning the region definedby SNP marker NN0228579 (map position 25.8), to SNP marker NN0224495(map position 82.0).

In certain embodiments, the DM resistance QTL of chromosome 5 is definedas spanning the region defined by SNP marker NN0228457 (map position25.2), to SNP marker NN0228148 (map position 82.7), as listed in Tables13 and 19. In other embodiments, the DM resistance QTL of chromosome 5is defined as spanning the region defined by SNP marker NN0224856 (mapposition 28.5) to SNP marker NN0223160 (map position 75.0). In yet otherembodiments, the DM resistance QTL of chromosome 5 is defined asspanning the region defined by SNP marker NN5096749 (map position 25.2),to SNP marker NN0227071 (map position 78.3).

QTL 1 has also been defined in a mapping population based on a cross ofcucumber lines Lucinde and PI197088, as being located between geneticmarkers ENK59 and CAPs_(—)17563/66, at about 5 cM-39 cM in the linkagegroup, and as defined by analysis of that mapping population (e.g. seeFIG. 1). Further, one of skill in the art would understand thatassignment of such genetic map positions may be affected by the mappingpopulation being analyzed, including for instance the parent lines used,the marker density, and the size of the population, each of which mayaffect the level of recombination which is seen, and thus the assignedgenetic map position. An integrated genetic and physical map may beutilized to define the position of a cucumber QTL, such as one providedby Ren et al, 2009, for instance relative to markers with known geneticand/or physical map positions.

The DM resistant cucumber plants of the present invention may bear oneor more alleles conferring DM resistance that have been introduced intothe cucumbers from a line designated PI197088 comprising the DMresistance, but otherwise comprising poor agronomic characteristics. Theresulting DM resistant cucumber plants of the present inventionsurprisingly display elite agronomic traits in combination with DMresistance, while lacking deleterious traits.

DM resistant cucumber plants may have large leaves that form a canopyover the fruit. The vine is typically indeterminate and grown ontrellises or the ground. DM resistant cucumber plants may have darkgreen, green, light green to yellow, and occasionally yellow to brownleaves. The leaves of the DM resistant cucumber plants vary in size, buttypically are from about 200-250 mm in length and 150-200 mm in width,and are usually simple, alternate, palmate, and lobed.

The ripe fruit of DM resistant cucumber plants of the present inventioncan vary from light to medium green, or even dark green, and typicallythe color on an individual fruit varies from a lighter-colored blossomend to a darker colored stem-end. The color may be mottled with yellowspeckles. The fruit of DM resistant cucumber is typically elongated andcylindrical with rounded or blunt ends, but may also be straight orcurved, and is usually 25-30 cm in length at harvest maturity, althoughthe fruit may be edible at 11-14 cm. The skin of the fruit is typicallysmooth, dull and thick; the skin may be tough or tender with a variednumber of tubercules. The flesh of the fruit is usually cream colored,with or without stripes, and has a bitter-free taste.

As used herein, a “susceptible control cucumber plant” refers to acucumber plant susceptible to Downy Mildew (DM susceptible) includingcommercially available and wild relatives of modern cucumber plants. Inone aspect, the control cucumber plant is the variety MARAM, SMR58,CORONA, or SPRINT 440. A “resistant control cucumber plant” may also beutilized when evaluating DM resistant cucumber varieties. In oneembodiment, such a control is a cucumber plant that is not susceptibleto DM, but is otherwise agriculturally undesirable, for example, varietyPI197088. Similarly, some controls may have intermediate resistance, forexample, controls with intermediate resistance to DM may be DMP21, GP14,LLP-1, ADEFEM, SWEETSLICE, or POINSETT 76. As described herein, acontrol cucumber line is grown under similar environmental conditions asthe comparative cucumber line, according to the present disclosure.

As used herein, a “hybrid cucumber plant” includes a plant resultingdirectly or indirectly from crosses between populations, breeds orcultivars within the species Cucumis sativus. “Hybrid cucumber plant” asused herein also refers to plants resulting directly or indirectly fromcrosses between different varieties or genotypes.

As used herein, a “female parent” refers to a cucumber plant that is therecipient of pollen from a male donor line, which pollen successfullypollinates an egg. A female parent can be any cucumber plant that is therecipient of pollen. Such female parents can be male sterile, forexample, because of genic male sterility, cytoplasmic male sterility, orbecause they have been subject to manual emasculation of the stamens.Genic or cytoplasmic male sterility can be manifested in differentmanners, such as sterile pollen, malformed or stamenless flowers,positional sterility, and functional sterility.

As used herein, “cytoplasmic male sterility” refers to plants that arenot usually capable of breeding from self-pollination, but are capableof breeding from cross-pollination.

As used herein, “linkage” is a phenomenon wherein alleles on the samechromosome tend to segregate together more often than expected by chanceif their transmission was independent.

As used herein, a “marker” is an indicator for the presence of at leastone phenotype, genotype, or polymorphism. Markers include, but are notlimited to, single nucleotide polymorphisms (SNPs), cleavable amplifiedpolymorphic sequences (CAPS), amplified fragment length polymorphisms(AFLPs), restriction fragment length polymorphisms (RFLPs), simplesequence repeats (SSRs), insertion(s)/deletion(s) (“INDEL”(s)),inter-simple sequence repeats (ISSR), and random amplified polymorphicDNA (RAPD) sequences. A marker is preferably inherited in codominantfashion (both alleles at a locus in a diploid heterozygote are readilydetectable), with no environmental variance component, i.e.,heritability of 1. A “nucleic acid marker” as used herein means anucleic acid molecule that is capable of being a marker for detecting apolymorphism, phenotype, or both associated with DM resistance.Stringent conditions for hybridization of a nucleic acid probe or primerto a marker sequence or a sequence flanking a marker sequence refers,for instance, to nucleic acid hybridization conditions of 5×SSC, 50%formamide, and 42° C. As used herein, “marker assay” means a method fordetecting a polymorphism at a particular locus using a particularmethod, e.g. measurement of at least one phenotype (such as a visuallydetectable trait, including disease resistance), restriction fragmentlength polymorphism (RFLP), single base extension, electrophoresis,sequence alignment, allelic specific oligonucleotide hybridization(ASO), random amplified polymorphic DNA (RAPD), microarray-basedtechnologies, PCR-based technologies, and nucleic acid sequencingtechnologies, etc.

As used herein, a “desirable trait” or “desirable traits” that may beintroduced into DM resistant cucumber plants by breeding may be directedto the cucumber fruit or the cucumber plant. Desirable traits to beintroduced into cucumber plants and cucumber fruit may be independentlyselected. Desirable cucumber fruit traits, e.g. as displayed byagronomically elite lines or cultivars, and that may be independentlyselected include, but are not limited to: fruit size, shape, color,surface appearance; seed number, seed size, locule number; pericarpthickness and toughness; taste, bitterness, the presence of tubercles,and shelf life. Desirable cucumber plant traits, e.g. as displayed byagronomically elite lines or cultivars, and that may be independentlyselected include, but are not limited to: plant vigor, leaf shape, leaflength, leaf color, plant height, whether the plant is determinate ornot, time to maturity, adaptation to field growth, adaptation togreenhouse growth, and resistance to one or more diseases or diseasecausing organisms such as Verticillium wilt, root knot nematodes,Tobacco Mosaic Virus, Cucumber scab, Anthracnose race 1, Powdery mildew(e.g. caused by Erysiphe cichoracearum or Sphaerotheca fuliginea),Target spot, Cucumber Mosaic Virus and Fusarium wilt. Any combination ofdesirable cucumber fruit traits, cucumber plant traits, or cucumberplant and fruit traits may be combined with a DM resistance trait. Theresulting agronomically elite DM resistant cucumber plants of thepresent invention surprisingly display such agronomic traits incombination with DM resistance, while lacking deleterious traits.

As used herein, “polymorphism” means the presence of one or morevariations of a nucleic acid sequence at one or more loci in apopulation of one or more individuals. The variation may comprise but isnot limited to one or more base changes, the insertion of one or morenucleotides or the deletion of one or more nucleotides. A polymorphismmay arise from random processes in nucleic acid replication, throughmutagenesis, as a result of mobile genomic elements, from copy numbervariation and during the process of meiosis, such as unequal crossingover, genome duplication and chromosome breaks and fusions. Thevariation can be commonly found, or may exist at low frequency within apopulation, the former having greater utility in general plant breedingand the latter may be associated with rare but important phenotypicvariation. Useful polymorphisms may include single nucleotidepolymorphisms (SNPs), insertions or deletions in DNA sequence (Indels),simple sequence repeats of DNA sequence (SSRs) a restriction fragmentlength polymorphism, and a tag SNP. A genetic marker, a gene, aDNA-derived sequence, a haplotype, a RNA-derived sequence, a promoter, a5′ untranslated region of a gene, a 3′ untranslated region of a gene,microRNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, dsRNA, atranscriptional profile, and a methylation pattern may comprisepolymorphisms. In addition, the presence, absence, or variation in copynumber of the preceding may comprise a polymorphism.

As used herein, “genotype” is the actual nucleic acid sequence at alocus in an individual plant. As used herein, “phenotype” means thedetectable characteristics (e.g. level of DM resistance) of a cell ororganism which can be influenced by genotype.

DM resistance of a cucumber plant provided herein can potentially bedefined as complete resistance or partial resistance. The DM resistanceof a cucumber plant provided herein can be measured by any meansavailable in the art.

In one aspect, DM resistance of a cucumber plant is determined by usinga disease rating of foliar chlorotic and/or necrotic lesion developmentafter inoculation or infection with DM on cucumber leaves using a scaleof symptoms of 0%, 10%, 20%, 30%, 40%, 50%, 60% and greater than about60% lesion covering the leaf area. A disease rating of 0% indicates acompletely resistant plant.

In another aspect, DM resistance is determined by obtaining diseaseratings of symptom development after one or more rounds of inoculationor infection with DM on cucumber leaves and/or cotyledons.

Resistance in a leaf test may be scored on an exemplary scale asfollows:

Index Value Symptoms 1 Absence of symptoms 2 Few small necrotic lesionswithout expansion 3 Few chlorotic and some necrotic lesions with limitedexpansion 4 Large expanding angular chlorosis with limited necroticlesions 5 Large expanding angular chlorosis with expanding necroticlesionsTests are evaluated once symptoms have developed on susceptible checks(e.g. cultivars Maram or SMR58). PI 197088 may be used as a “resistant”control; and cv. Poinsett 76 or other cv. exhibiting a comparable levelof Downy Mildew resistance may be used as a control to assess“intermediate” levels of resistance/susceptibility to P. cubensis. Threeobservations are made on each plot, one at each end and one in themiddle. The mean disease index for each plot is calculated. These areaveraged for all three replicates and the standard deviation isdetermined. The disease index ranges for the categories “Resistant,”“Intermediate Resistant” and “Susceptible” are then determined.Varieties are generally trialed several times before a final diseaseresistance level determination is made. Scores of 1-5 indicate varyinglevels of resistance or susceptibility. A score of 1-2 after one or morerounds of inoculation or infection, and preferably two or more rounds ofinfection, indicates a resistant plant. A score of 3 after one or morerounds of inoculation or infection, preferably two or more rounds ofinfection, indicates a plant exhibiting intermediate resistance. A scoreof 4-5 indicates a susceptible plant. Scores on this 1-5 scale wouldcorrelate to a 1-9 scale where 1=1, 2=3, 3=5, 4=7, and 5=9. The degreeof resistance may also be assessed by an alternative rating scale, forinstance as described in Example 6.

In one aspect of the invention, a plant is assayed for DM resistance,partial resistance or susceptibility by image analysis of foliar tissueusing about 3 leaves per plant captured in a digital image. The imageanalysis is conducted to determine the percentage of tissue damage andderive a disease rating. Image analysis software and methods used forquantifying visual differences in two or three dimensions are those setforth in Bright, 1987 (J. Microscopy 148:51-87) and Bickmore et al.,1999 (Geol. Mat. Res. 1(5):1-19). With respect to image analysis: “veryresistant” exhibits between about 0% and 5% leaf area symptoms ofchlorotic and/or necrotic lesions; “resistant” is between about 1% and20% of the leaf area having symptoms of chlorotic and/or necroticlesions; “substantially resistant” is between about 20% and 30% of theleaf area having symptoms of chlorotic and/or necrotic lesions;“mid-resistant” is between 40% and 50% of the leaf area having symptomsof chlorotic and/or necrotic lesions; “partially resistant” is less thanor equal to about 50% of the leaf area having symptoms of chloroticand/or necrotic lesions; “mid-susceptible” is between about 50% and 60%of the leaf area having symptoms of chlorotic and/or necrotic lesions;and “susceptible” is between about 60% and 100% of the leaf area havingsymptoms of chlorotic and/or necrotic lesions. A resistant plant can becharacterized by other aspects as set forth herein, or by the use ofother means, such as quantitative PCR to determine the level ofinfection.

Cucumber lines having DM resistance, or partial resistance, demonstratea reduced level of symptoms relative to a non-resistant control cucumberline after inoculation or infection with DM. The level of symptoms canbe used as an indicator of DM resistance. Disease symptoms measured canbe any disease symptoms associated with DM infection. Symptoms can beselected from the group consisting of leaf blisters, necrosis, softfruits, mosaic, chlorotic veins, chlorotic leaf spots, chlorotic and/orlight green mosaic on leaves, fruit lesions, or combinations thereof. Inone aspect, a DM resistant cucumber line demonstrates a reduction offoliar symptoms of chlorotic and/or necrotic lesions of at least, orgreater than, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, or 98% relative to a non-resistant control cucumber line. Inother aspects, the leaves of a DM resistant cucumber plant demonstrateless than 15%, or less than 10%, or less than 5%, or less than 2%symptomatic area when exposed to DM. In another aspect, the cucumberplant belongs to a cucumber variety or cultivar, and in another aspect,the cucumber plant is an inbred cucumber plant.

In another aspect, the cucumber plants and varieties provided hereindemonstrate little or no symptoms of chlorotic and/or necrotic lesionsafter inoculation or infection with DM. In some aspects, a DM resistantcucumber plant demonstrates symptoms of chlorotic and/or necroticlesions on less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% 2%, or 1% of thecucumber leaf surface.

DM resistant cucumber plants may exhibit a delay in the onset ofsymptoms of chlorotic and/or necrotic lesions relative to anon-resistant control cucumber plant. In some embodiments, the DMresistant cucumber plants exhibit a delay of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or more days in the onset of symptoms of chloroticand/or necrotic lesions relative to a control cucumber plant. In otherembodiments, the DM resistant cucumber plants exhibit a delay of atleast 7 or more days, 10 or more days, or 14 or more days in the onsetof symptoms of chlorotic and/or necrotic lesions relative to a controlcucumber plant.

In one aspect, the cucumber plant is a seedling at the time ofinoculation or infection. In some aspects, the cucumber plant is aseedling at the 4, 5, 6, 7, or 8 leaf stage of development wheninoculated. In one aspect, disease symptoms can be measured at any timeafter pathogenic challenge of a cucumber plant. In other aspects,symptoms can be measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or more days after inoculation. In anotheraspect, the cucumber plant is any age of plant at the time ofinoculation or infection.

In another aspect, disease symptoms can be observed after DM challengeof an entire plant or a part thereof, for example, a plant cutting.

DM resistant cucumber plants of the present invention may exhibit anincrease in fruit yield after inoculation or infection with DM relativeto a control cucumber plant inoculated with DM. In one aspect, theresistant cucumber plants exhibit a 2%, 5%, 10%, 15%, 20% or moreincrease in fruit yield, based upon the total mass, number, or totalvolume of fruit, relative to a control cucumber plant after one or morerounds of inoculation or infection with DM.

The present invention provides for and includes cucumber plants thatexhibit resistance to one or more races of DM. In some embodiments, thecucumber plants of the present invention exhibit resistance to 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more races of DM.

The present invention provides for a seed of a cucumber plant capable ofproducing a plant having DM resistance. In one aspect, the cucumberplant can be an open-pollinated variety, a hybrid parent inbred line, ora male sterile line. In another aspect, the invention provides seed of acucumber plant capable of producing a hybrid cucumber plant having DMresistance.

The cucumber plants of the present invention can be cucumber linesadapted for greenhouse cucumber production or for field cucumberproduction. In one aspect, the cucumber plants of the present inventionare adapted for greenhouse cucumber production.

The present invention also provides a hybrid cucumber having DMresistance. In another aspect, the present invention provides a hybridcucumber exhibiting DM resistance after inoculation or infection withDM.

Commercially valuable cucumber plants represent one aspect of thepresent invention. In one aspect, certain cucumber traits, including,for example, fruit size, shape, color, weight, taste and fruit yield maybe important to the commercial value of the crop. Fruit size, and shape,may be of particular interest if the cucumbers are grown for processingsuch as pickling. The present invention provides for a cucumber plantthat produces a cucumber fruit having a length of about, or greater thanabout, 11, 12, 13, or 14 cm. In another aspect, a cucumber plant of thepresent invention produces a cucumber fruit having a length betweenabout 11 and 13 cm, 12 and 14 cm, and 11 and 14 cm.

In some aspects, a cucumber plant of the present invention may produce acucumber fruit having a weight at harvest of about or greater than about80, 85, 90, 95, 100, 105, 110, 115, 120, and 125 grams. In otheraspects, a cucumber plant of the present invention produces a cucumberfruit having a weight at harvest between about 80 and about 125 grams,about 90 and about 115 grams, about 100 and about 120 grams, about 90and about 125 grams, about 95 and about 125 grams, about 100 and about125 grams, or between about 115 and about 125 grams. Fruit weight ismeasured by weighing individual cucumber fruit on a scale.

Mature cucumber fruit produced by DM resistant plants of the presentinvention may have a diameter from about 10, 11, 12, 13, or 14 mm orlarger. In some embodiments the diameter of the cucumber fruit may befrom about 10 to about 11 mm, or from about 10 to about 12 mm, or fromabout 11 to about 13 mm, or from about 12 to about 14 mm, or from about13 to about 14 mm.

A cucumber fruit attribute such as shape, weight, or size can bemeasured or evaluated at a variety of times. In one aspect, an attributeis measured following growth in a growth chamber. In another aspect, anattribute is measured at the time of harvest. In yet another aspect, anattribute is measured after storage of the cucumber fruit at ambientconditions for one day, two days, three days, four days, five days, sixdays, seven days, eight days, nine days, ten days, eleven days, twelvedays, thirteen days, two weeks, three weeks, four weeks, or five weeksafter harvest.

In one embodiment, a cucumber fruit from a cucumber plant having DMresistance has an overall fruit quality rating of 1, 3, 5, 7, or 9,where fruit quality is measured by visual inspection, with a scaleranging from 1=excellent through 9=poor: Rating 1=Excellent; 3=Aboveaverage; 5=Average; 7=Below average; 9=Poor; compared to the standardcommercial hybrids grown in the area. Fruit Quality relates to fruitcolor, fruit shape, fruit length and diameter.

A further aspect of the invention relates to tissue cultures of thecucumber plants described herein. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of one ormore types, or a collection of such cells organized into parts of aplant. Tissue culture includes, but is not limited to, compositionscomprising protoplasts and calli. Tissue culture also includes, but isnot limited to, compositions comprising plant cells that are present inintact plant tissues, or parts of plants, such as embryo, leaf,peduncle, pedicel, anther, meristem, tip and segments of root, stump andstem, explants, and the like. In one aspect, a tissue culture comprisesembryos, protoplasts, meristematic cells, pollen, leaves, anthers orcells derived from immature tissues of these plant parts. Means forpreparing and maintaining plant tissue cultures are well known in theart. Examples of processes of tissue culturing and regeneration ofcucumber are described in, for example, Fillatti et al., 1987(Bio/Technology, 5:726-730). In some aspects, tissue culture of thecucumber plants described herein relates to the culture of protoplasts,calli, or plant cells, that are isolated from, or present in, intactparts of the DM resistant plants described herein. In another aspect,tissue culture refers to the culture of protoplasts, calli, or plantcells, that are isolated from, or present in, intact parts of plants ofone or more DM resistant cucumber plant lines selected from the groupconsisting of ASL147-2027, EUR154-1012GY, EUR154-1021GY, GSP33-1094GY,GPN33-1093GY, 03/8020-20_TUP03_DMFL_(—)1, 03/8024-19_TUP03_DMFL_(—)1,and 03/8039-5_TUP03_DMFL_(—)1, and DM resistant progeny thereof,including those produced by crosses or backcrosses, as listed in U.S.patent application Ser. No. 12/424,452, published as US 2009-0265803,the entire disclosure of which is incorporated herein by reference.Representative samples of seed of said lines have been deposited underATCC Accession Number PTA-9375, ATCC Accession Number PTA-8930, ATCCAccession Number PTA-8931, ATCC Accession Number PTA-8953, ATCCAccession Number PTA-8954, ATCC Accession Number ______, ATCC AccessionNumber ______, and ATCC Accession Number ______, respectively, and DMresistant progeny thereof, as listed in U.S. Patent publication2009-0265803.

In yet another aspect, tissue culture of the cucumber plants describedherein relates to the culture of protoplasts, calli, or plant cells,that are isolated from, or present in, intact parts of the DM resistantplants described herein.

Once DM resistant plants are produced, the plants themselves can becultivated in accordance with conventional procedures. DM resistantprogeny may be obtained through sexual reproduction. The seeds resultingfrom sexual reproduction can be recovered from the fruit of DM resistantplants and planted or otherwise grown as a means of propagation. DMresistant progeny may also be obtained from DM resistant plants throughasexual reproduction. Protoplast or propagules (e.g., cuttings, scionsor rootstocks) can be recovered from DM resistant plants or partsthereof and may be employed to propagate DM resistant plants.

The present invention also provides for and includes a container ofcucumber seeds in which cucumber plants grown from greater than 50% ofthe seeds have resistance or partial resistance to DM. In anotheraspect, cucumber plants grown from greater than 55%, 65%, 75%, 85%, 90%,95%, 98%, or 99% of the cucumber seeds in the container have DMresistance. Another aspect of the invention relates to seeds from acucumber plant selected from the group consisting of: ASL147-2027,EUR154-1012GY, EUR154-1021GY, GSP33-1094GY, GPN33-1093GY,03/8020-20_TUP03_DMFL_(—)1, 03/8024-19_TUP03_DMFL_(—)1, and03/8039-5_TUP03_DMFL_(—)1, and DM resistant progeny thereof, whereincucumber plants grown from about 50%, or greater than 50%, of the seedshave resistance or partial resistance to DM.

The container of cucumber seeds can contain any number, weight or volumeof seeds. For example, a container can contain about, or greater thanabout, 10, 25, 50, 200, 400, 700, 1000, 2000, 3000, or more seeds. Inanother aspect, a container can contain about, or greater than about, 1gram, 5, 10, 15, 25, 100, 250, 500, or 1,000 grams of seeds.Alternatively, the container can contain about or at least, or greaterthan, about 1 ounce, 2, 4, 8, 10 ounces, 1 pound, 2, 4, 8, 12 pounds ormore of seeds.

Containers of cucumber seeds can be any container available in the art.For example, a container can be a box, a bag, a packet, a pouch, a taperoll, a foil, a pail, or a tube.

The present invention includes and provides for a container of cucumberfruit from cucumber plants having DM resistance. In one aspect, thecontainer contains about 2, 5, 10, 20, 40, 80, 100, or more cucumberfruit. In yet another aspect, the present invention provides a cucumbervine having cucumber fruit from a plant having resistance to DM.

One aspect of the invention relates to dried, or otherwise processed,cucumber fruit, produced by a cucumber plant having a genome thatcomprises at least one genetic locus giving rise to DM resistance whenexpressed in a cucumber plant. Processed cucumber fruit includes, but isnot limited to fruit pulp, stewed cucumbers, canned, pickled, minced,sliced, or crushed cucumber fruit. In some aspects, the dried, pickled,or otherwise processed cucumber fruit, is the fruit of a cucumber plantof a line selected from the group consisting of: ASL147-2027,EUR154-1012GY, EUR154-1021GY, GSP33-1094GY, GPN33-1093GY,03/8020-20_TUP03_DMFL_(—)1, 03/8024-19_TUP03_DMFL_(—)1, and03/8039-5_TUP03_DMFL_(—)1.

The present invention provides for an inbred cucumber plant havingresistance to DM, wherein the resistance is exhibited when the plant isin contact with DM. In one aspect, the inbred cucumber plant is derivedfrom accession PI197088.

The present invention includes and provides for C. sativus plants havingat least one allele for a DM resistance trait. The DM resistant cucumberplants can be either heterozygous or homozygous for the DM resistancetrait. In one embodiment, the DM resistant trait can be linked tovariations in a single gene (e.g., linked to one or more alleles of asingle gene). In another embodiment, the DM resistance trait can belinked to variations at one or one or more quantitative trait loci(QTL). In a yet another embodiment, the DM resistant cucumber plants arehomozygous for the DM resistance trait.

The present invention provides for a C. sativus cucumber plant having agenome that comprises at least one genetic locus that provides DMresistance from a non-C. sativus plant. In some aspects, the DMresistant cucumber plant is selected from the group consisting of:ASL147-2027, EUR154-1012GY, EUR154-1021GY, GSP33-1094GY, GPN33-1093GY,03/8020-20_TUP03_DMFL_(—)1, 03/8024-19_TUP03_DMFL_(—)1, and03/8039-5_TUP03_DMFL_(—)1, and DM resistant progeny thereof. In oneaspect, the genetic locus derived from a DM resistant cucumber plant canbe identified using genetic markers.

The present invention provides for a DM resistant C. sativus cucumberplant having less than or equal to 50% of its genome derived from anon-C. sativus DM resistant plant. In another aspect, a DM resistant C.sativus cucumber plant can have 50%, 25%, 12.5%, 6%, 3% or less nuclearDNA derived from a DM resistant non-C. sativus plant. In other aspects,a DM resistant C. sativus cucumber plant can have 50%, 25%, 12.5%, 6% or3% or less nuclear DNA derived from another member of the Cucumis genusthat is DM resistant.

The present invention provides progeny of cucumber plants havingresistance to DM. As used herein, progeny include not only, withoutlimitation, the products of any cross (be it a backcross or otherwise)between two plants, but all progeny whose pedigree traces back to theoriginal cross. In one aspect of the present invention, the progenycontain about 50%, 25%, 12.5% or less nuclear DNA from a DM resistantcucumber plant and expresses the genetic material that provides DMresistance. Representative populations of cucumber plants comprisingprogeny having resistance to DM include progeny of the cross ofsusceptible parent cv. Lucinde×PI197088, as well as the API and VJpopulations, generated from crossing each of two susceptible parents(API-45-5312-MO and VJ03-68-06) by the same resistant line (PI197088).

One embodiment of the present invention provides for a DM resistantcucumber plant that contains a genetic marker linked to one or more DMresistance locus. By “DM resistance locus” is meant a locus thatcontributes to DM resistance either alone or in combination with onemore other DM resistance locus. By “contributes to Downy Mildewresistance” it is meant that the degree of Downy Mildew resistance isincreased in the corresponding plant, either when the locus is alone orin combination with one or more other locus.

In one embodiment of the invention, a marker linked to one or more DMresistance loci includes one or more of the following: CAPs_(—)21826,CAPs_ENK60, CAPs_ENK59, CAPs_(—)17170, CAPs_(—)17179, CAPs_(—)18229CAPs_(—)17563/66, and CAPs_ENK70. In another embodiment of theinvention, the assayed markers linked to one or more DM resistance lociinclude each of the following: CAPs_ENK60, CAPs_(—)17170, andCAPs_(—)17563/66. In yet another embodiment, the marker(s) linked to oneor more DM resistance loci includes one or more SNP marker(s) selectedfrom the group consisting of: NN0223782, NN0225385, NN0226670,NN0224124, NN0246472, NN0225358, NN0227700, NN0224617, NN0247695,NN0227242, NN0223824, NN0223181, NN0226638, NN0225012, NN0228579,NN0226451, NN0225088, NN0226219, NN0247551, NN0246357, NN0225551,NN0226732, NN0247689, NN0247342, NN0224702, NN0225482, NN0224538,NN0247543, NN0224041, NN0228853, NN0227762, NN0227587, NN0228457,NN0246356, NN0246332, NN0223399, NN0223689, NN0247731, NN0226166,NN0225480, NN0246411, NN0227759, NN0247348, NN0228465, NN0247786,NN0226645, NN0223809, NN0227071, NN0226870, NN0228148, NN0246378,NN0224041, NN0227587, NN5096749, NN0224856, NN0223160, NN0223809,NN0226631, NN0227981, NN0247786, NN0246425, NN0224495, NN0225088,NN0227071, NN0223782, NN0228579, NN0247342, and NN0247731, comprising asingle nucleotide polymorphism of one of SEQ ID NOs:20-87.

As used herein, linkage of two nucleic acid sequences, including anucleic acid marker sequence and a nucleic acid sequence of a geneticlocus imparting a desired trait such as DM resistance, may be genetic orphysical or both. In one aspect of the invention, the nucleic acidmarker and genetic locus conferring DM resistance are geneticallylinked, and exhibit a LOD score of greater than 2.0, as judged byinterval mapping for the DM resistance trait based on maximum likelihoodmethods described by Lander and Botstein, 1989 (Genetics, 121:185-199),and implemented in the software package MAPMAKER (e.g. Lander et al.,Genomics 1:174-181, (1987); default parameters). Alternatively, othersoftware such as QTL Cartographer v1.17 (Basten et al., Zmap—a QTLcartographer. In: Proceedings of the 5th World Congress on GeneticsApplied to Livestock Production: Computing Strategies and Software,edited by C. Smith, J. S. Gavora, B. Benkel, J. Chesnais, W. Fairfull,J. P. Gibson, B. W. Kennedy and E. B. Burnside. Volume 22, pages 65-66.Organizing Committee, 5th World Congress on Genetics Applied toLivestock Production, Guelph, Ontario, Canada, 1994; and Basten et al.,QTL Cartographer, Version 1.17. Department of Statistics, North CarolinaState University, Raleigh, N.C., 2004) may be used. Mapping of QTLs iswell-described (e.g. WO 90/04651; U.S. Pat. Nos. 5,492,547, 5,981,832,6,455,758; reviewed in Flint-Garcia et al. 2003 (Ann. Rev. Plant Biol.54:357-374, the disclosures of which are hereby incorporated byreference). In other embodiments, the marker and region conferring DMresistance are genetically linked and exhibit a LOD score of greaterthan 3.0, or a LOD score of greater than 6.0, 9.0, 12.0, 15.0, or 18.0.In one embodiment, the marker and region contributing to DM resistanceare genetically linked and exhibit a LOD score of between about 14 andabout 20. When assigning the presence of a QTL, the LOD threshold scoreassociated with a QTL analysis as described herein may be determined tobe significant at the 95% confidence level, or higher, such as at the98% or 99% confidence level.

In another aspect, the nucleic acid marker is genetically linked at adistance of between about 0 and about 50 centimorgans (cM) to the DMresistance locus. In other embodiments, the distance between the nucleicacid marker and the DM resistance locus is between about 0 and about 35cM, or between about 0 and about 25 cM, or between about 0 and about 15cM, or between about 0 and about 10 cM, or between about 0 and about 5cM, including less than about 4, 3, 2 or 1 cM.

In yet another aspect, the invention provides a cucumber plantcomprising an introgressed chromosomal region from chromosome 2 ofPI197088 or a progeny plant thereof, of 20 cM, 10 cM, 5 cM, or 1 cMwithin the region defined as spanning the positions of SNP markerNN0246378 and SNP marker NN0246472; or SNP marker NN0246378 and SNPmarker NN0247695. In still yet another aspect, the invention provided acucumber plant comprising an introgressed chromosomal region fromchromosome 4 of PI197088 or a progeny plant thereof, of 20 cM, 10 cM, 5cM, or 1 cM within the region defined as spanning the positions of SNPmarker NN0225012 and SNP marker NN0227587; or SNP marker NN0228579 andSNP marker NN0224495; or SNP marker NN0225088 and SNP marker NN224041.In yet another aspect, the invention provides a cucumber plantcomprising an introgressed chromosomal region from chromosome 5 ofPI197088 or a progeny plant thereof, of 20 cM, 10 cM, 5 cM, or 1 cMwithin the region defined spanning the positions of SNP marker NN5096749and NN0227071; or SNP marker NN5096749 and SNP marker NN0223160; or SNPmarker NN0224856 and SNP marker NN0223160. In still other embodiments,the cucumber plant may comprise an introgressed chromosomal region ofchromosome 2 from PI197088 and an introgressed chromosomal region ofchromosome 4 or 5 of PI197088, wherein the introgressed chromosomalregions allow for enhanced resistance to DM, relative to an otherwiseisogenic cucumber line not comprising one or more of the introgressedregion(s).

Further, the cucumber plant may comprise an introgressed chromosomalregion of chromosome 4 from PI197088 and an introgressed chromosomalregion of chromosome 2 and/or 5 of PI197088, or an introgressed regionfrom chromosome 5 of PI197088 or a progeny thereof, and an introgressedchromosomal region of chromosomes 2 and/or 4 of PI197088 or a progenyplant thereof, wherein the introgressed chromosomal region(s) allow forenhanced resistance to DM, relative to an otherwise isogenic cucumberline not comprising one or more of the introgressed region(s).

In particular embodiments, the introgressed chromosomal fragment fromPI197088 or a progeny plant thereof, comprises a chromosomal region ofabout 20 cM, 10, cM, 5 cM, or 1 cM, and further comprises aPI197088-derived allele at SNP marker NN0226631 of chromosome 5. In yetother embodiments, the introgressed chromosomal fragment from PI197088or a progeny plant thereof, comprises a chromosomal region of about 20cM, 10, cM, 5 cM, or 1 cM, and further comprises a PI197088-derivedallele at SNP marker NN0246425 of chromosome 4.

In another aspect, the nucleic acid marker sequence may be physicallylinked to a DM resistance locus. In some aspects, the nucleic acidsequence of the genetic marker specifically hybridizes to a nucleic acidmolecule having a sequence that is within about 30 Mbp, or about 20 Mbp,or about 15 Mbp, or about 10 Mbp, or about 5 Mbp of a DM resistancelocus. In another aspect, the nucleic acid sequence of the geneticmarker specifically hybridizes to a nucleic acid molecule having asequence of any of SEQ ID NOs:1-87, or a complement thereof.

As used herein, two nucleic acid molecules are said to be capable ofhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. Conventionalstringency conditions are described by Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989) and by Haymes et al., Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. Thus, in order for anucleic acid molecule to serve as a primer or probe it need only besufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. In some embodiments,hybridization conditions can be high, moderate or low stringencyconditions. Preferred conditions include those using 50% formamide,5.0×SSC, 1% SDS and incubation at 42° C. for 14 hours, followed by awash using 0.2×SSC, 1% SDS and incubation at 65° C.

The specificity of hybridization can be affected by post-hybridizationwashes. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a moderatestringency of about 1.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to moderate stringency conditions at about 50° C., to highstringency conditions at about 65° C. Both temperature and saltconcentration may be varied, or either the temperature or the saltconcentration may be held constant while the other variable is changed.In some aspects, the wash step can be performed for 5, 10, 15, 20, 25,30, or more minutes. In another aspect, the wash step is performed forabout 20 minutes. In yet another aspect, the wash step can be repeated1, 2, 3, 4, or more times using the selected salt concentration,temperature, and time. In another aspect, the wash step is repeatedtwice.

A genetic marker profile of a plant may be predictive of the agronomictraits of a hybrid produced using that inbred. For example, if an inbredplant of known genetic marker profile and phenotype is crossed with asecond inbred of known genetic marker profile and phenotype it ispossible to predict the phenotype of the F₁ hybrid based on the combinedgenetic marker profiles of the parent inbreds. Methods for prediction ofhybrid performance from genetic marker data are disclosed in U.S. Pat.No. 5,492,547, the disclosure of which is specifically incorporatedherein by reference in its entirety. Such predictions may be made usingany suitable genetic marker, for example, SSRs, INDELs, RFLPs, AFLPs,SNPs, ISSRs, or isozymes.

Additional markers, such as SSRs, AFLP markers, RFLP markers, RAPDmarkers, phenotypic markers, SNPs, isozyme markers, or microarraytranscription profiles that are genetically linked to or correlated withDM resistance can be utilized (Walton, Seed World 22-29 (July, 1993);Burow and Blake, Molecular Dissection of Complex Traits, 13-29, Eds.Paterson, CRC Press, New York (1988)). Methods to isolate such markersand to design probes or primers useful in following the presence of suchmarkers are known in the art. For example, locus-specific SSRs can beobtained by screening a cucumber genomic library for SSRs, sequencing of“positive” clones, designing primers which flank the repeats, andamplifying genomic DNA with these primers. Likewise, SNP markers may beidentified as well.

The genetic linkage of marker molecules to DM resistance can beestablished by a gene mapping model such as, without limitation, theflanking marker model, and the interval mapping, based on maximumlikelihood methods described by Lander and Botstein, 1989 (Genetics,121:185-199), and implemented in the software packages MAPMAKER(Whitehead Institute for Biomedical Research, Cambridge Mass., USA) orQTL Cartographer (North Carolina State University, BioinformaticsResearch Center) or the like.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no trait effect, to avoidfalse positives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a trait (MLE given no linked trait)).

The LOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a resistance allele rather than inits absence. The LOD threshold value for avoiding a false positive witha given confidence, say 95%, depends on the number of markers and thelength of the genome. Graphs indicating LOD thresholds are set forth inLander and Botstein (1989), and further described by Ars andMoreno-Gonzalez, Plant Breeding, Hayward, Bosemark, Romagosa (eds.)Chapman & Hall, London, pp. 314-331 (1993), and van Ooijen (Heredity83:613-624, 1999).

Selection of appropriate mapping or segregation populations is importantin trait mapping. The choice of appropriate mapping population dependson the type of marker systems employed (Tanksley et al., Molecularmapping plant chromosomes. Chromosome structure and function: Impact ofnew concepts J. P. Gustafson and R. Appels (eds.), Plenum Press, NewYork, pp. 157-173 (1988)). Consideration must be given to the source ofparents (adapted vs. exotic) used in the mapping population. Chromosomepairing and recombination rates can be severely disturbed (suppressed)in wide crosses (adapted x exotic) and generally yield greatly reducedlinkage distances. Wide crosses will usually provide segregatingpopulations with a relatively large array of polymorphisms when comparedto progeny in a narrow cross (adapted×adapted).

Advanced breeding lines are collected from breeding programs. These aretested for their phenotype (e.g. their disease score reactions to DM),and genotyped for markers in the DM QTL intervals. From these data, thesmallest genetic interval is identified within each QTL containing thedonor parent (DP) favorable allele among the DM resistant lines. Thisinterval is inferred to be critical for conferring resistance to DM.Candidate genetic intervals associated with DM resistance were detectedas regions showing enhanced frequency of the favorable allele from theDM resistance donor PI197088 relative to a baseline set of DMsusceptible samples from the same germplasm classification type (GCT).For example, comparisons may be made among DM resistant and susceptibleinbreds within a single GCT and a single breeding program. Allelefrequency shifts between phenotypic classes may be detected bycalculating a linkage assessment score (LAS) as: LAS=(Frequency offavorable allele in samples with favorable phenotype)×(Frequency ofunfavorable allele in samples with unfavorable phenotype).

As used herein, the progeny include not only, without limitation, theproducts of any cross (be it a backcross or otherwise) between twoplants, but all progeny whose pedigree traces back to the originalcross. Specifically, without limitation, such progeny include plantsthat have 50%, 25%, 12.5% or less nuclear DNA derived from one of thetwo originally crossed plants. As used herein, a second plant is derivedfrom a first plant if the second plant's pedigree includes the firstplant.

The present invention provides a genetic complement of the cucumberlines described herein. Further provided is a hybrid genetic complement,wherein the complement is formed by the combination of a haploid geneticcomplement from elite inbred cucumber lines described herein and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well-known in the art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which defines the phenotype of aplant, such as a C. sativus cucumber plant or a cell or tissue of thatplant. By way of example, a C. sativus cucumber plant is genotyped todetermine a representative sample of the inherited markers it possesses.Markers are preferably inherited in codominant fashion so that thepresence of both alleles at a diploid locus is readily detectable, andthey are free of environmental variation, i.e., their heritability isclose to, or equal to, 1. This genotyping is preferably performed on atleast one generation of the descendant plant for which the numericalvalue of the trait or traits of interest are also determined. The arrayof single locus genotypes is expressed as a profile of marker alleles,two at each locus for a diploid plant. The marker allelic composition ofeach locus can be either homozygous or heterozygous. Homozygosity is acondition where both alleles at a locus are characterized by the sameconditions of the genome at a locus (e.g., the same nucleotidesequence). Heterozygosity refers to different conditions of the genomeat a locus. Potentially any type of genetic marker could be used, forexample, simple sequence repeats (SSRs), insertion/deletion polymorphism(INDEL), restriction fragment length polymorphisms (RFLPs), amplifiedfragment length polymorphisms (AFLPs), single nucleotide polymorphisms(SNPs), and isozymes.

Considerable genetic information can be obtained from a completelyclassified F₂ population using a codominant marker system (Mather,Measurement of Linkage in Heredity: Methuen and Co., (1938)). An F₂population is the first generation of self or sib pollination after thehybrid seed is produced. Usually a single F₁ plant is self or sibpollinated to generate a population segregating for the nuclear-encodedgenes in a Mendelian (1:2:1) fashion.

In contrast to the use of codominant markers, using dominant markersoften requires progeny tests (e.g., F₃ or back cross self families) toidentify heterozygous individuals. The information gathered can beequivalent to that obtained in a completely classified F₂ population.This procedure is, however, often prohibitive because of the cost andtime involved in progeny testing. Progeny testing of F₂ individuals isoften used in map construction where error is associated with singleplant phenotyping, or when sampling the plants for genotyping affectsthe ability to perform accurate phenotyping, or where trait expressionis controlled by a QTL. Segregation data from progeny test populations(e.g., F₃ or backcrossed or selfed families) can be used in traitmapping. Marker-assisted selection can then be applied to subsequentprogeny based on marker-trait map associations (F₂, F₃), where linkagehas not been completely disassociated by recombination events (i.e.,maximum disequilibrium).

Recombinant inbred lines (RILs) (genetically related lines; usually >F₅)can be used as a mapping population. RILs can be developed by selfing F2plants, then selfing the resultant F3 plants, and repeating thisgenerational selfing process, thereby increasing homozygosity.Information obtained from dominant markers can be maximized by usingRILs because all loci are homozygous or nearly so. Under conditions oftight linkage (i.e., about <10% recombination), dominant and co-dominantmarkers evaluated in RIL populations provide more information perindividual than either marker type in backcross populations (e.g. Reiteret al., 1992; Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481). However, asthe distance between markers becomes larger (i.e., loci become moreindependent), the information in RIL populations decreases dramaticallywhen compared to codominant markers.

Backcross populations can be utilized as mapping populations. Abackcross population (BC) can be created by crossing an F₁ to one of itsparents. Typically, backcross populations are created to recover thedesirable traits (which may include most of the genes) from one of therecurrent parental (the parent that is employed in the backcrosses)while adding one or a few traits from the second parental, which isoften referred to as the donor. A series of backcrosses to the recurrentparent can be made to recover most of the recurrent parent's desirabletraits. Thus a population is created consisting of individuals nearlylike the recurrent parent, wherein each individual carries varyingamounts or a mosaic of genomic regions from the donor parent. Backcrosspopulations can be useful for mapping dominant markers particularly ifall loci in the recurrent parent are homozygous and the donor andrecurrent parent have contrasting polymorphic marker alleles (Reiter etal., 1992; Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481).

Information obtained from backcross populations using either codominantor dominant markers is less than that obtained from completelyclassified F₂ populations because recombination events involving one,rather than two, gametes are sampled per plant. Backcross populations,however, are more informative (at low marker saturation) when comparedto RILs as the distance between linked loci increases in RIL populations(i.e., about 15% recombination). Increased recombination can bebeneficial for resolution of tight linkages, but may be undesirable inthe construction of maps with low marker saturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of theloci polymorphic between the parentals are expected to segregate in thehighly homozygous NIL population. Those loci that are polymorphic in aNIL population, however, are likely to be linked to the trait ofinterest.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore, et al., 1991; Proc. Natl. Acad. Sci. (U.S.A.)88:9828-9832). In BSA, two bulk DNA samples are drawn from a segregatingpopulation originating from a single cross. These bulk samples containindividuals that are identical for a particular trait (e.g., resistantor susceptible to a particular pathogen) or genomic region but arbitraryat unlinked regions (i.e., heterozygous). Regions unlinked to the targettrait will not differ between the bulked samples of many individuals inBSA.

In another aspect, the present invention provides a method of producinga DM resistant cucumber plant comprising: (a) crossing a cucumber linehaving DM resistance with a second cucumber line lacking DM resistanceto form a segregating population; (b) screening the population forresistance to DM; and (c) selecting one or more members of thepopulation having said DM resistance. In one aspect, the cucumber linehaving DM resistance is crossed with the second cucumber line for atleast two generations (e.g., creating either an F₂ or BC₁S₁ population).In a particular embodiment, the cucumber line having DM resistance isPI197088, or a progeny thereof. In another aspect, plants are identifiedas DM resistant prior to crossing. In one aspect, plants can be selectedon the basis of partial or complete resistance to DM. In one aspect, thesegregating population is self-crossed and the subsequent population isscreened for resistance.

In another aspect, the present invention provides a method ofintrogressing DM resistance into a cucumber plant comprising: (a)crossing at least a first cucumber line having DM resistance with asecond cucumber line to form a segregating population; (b) screeningsaid population for resistance to DM; and (c) selecting at least onemember of said population exhibiting DM resistance. In one aspect, thecucumber line having DM resistance is crossed with the second cucumberline for at least two generations (e.g., creating either an F₂ or BC₁S₁population), or up to 2-10 generations. In another aspect, plants areidentified as DM resistant prior to crossing. In one aspect, thesegregating population is self-crossed and the subsequent population isscreened for resistance.

Cucumber plants (and parts thereof, including seed, pollen, and ovules)generated using a method of the present invention are also provided, andcan be part of or generated from a breeding program. The choice ofbreeding method depends on the mode of plant reproduction, theheritability of the trait(s) being improved, and the type of cultivarused commercially (e.g., F₁ hybrid cultivar, pure line cultivar, etc).Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection of the progeny of any cross. It is furtherunderstood that any commercial and non-commercial cultivars can beutilized in a breeding program. Factors such as, for example, emergencevigor, vegetative vigor, stress tolerance, disease resistance,branching, flowering, fruit size, fruit quality, and/or fruit yield willgenerally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on statistical analyses(e.g., mean values) obtained from replicated evaluations of families ofrelated plants. Popular selection methods commonly include pedigreeselection, modified pedigree selection, mass selection, and recurrentselection. In a preferred embodiment a backcross or recurrent breedingprogram is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates as parents for newcommercial cultivars; those still deficient in traits may be used asparents for hybrids, or to produce new populations for furtherselection.

One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated self or sib pollinating and selection, producing many newgenetic combinations.

The development of new cucumber lines requires the development andselection of cucumber varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Hybrids can be selected for certain single genetraits such as flower color, seed yield or herbicide resistance thatindicate that the seed is truly a hybrid. Additional data on parentallines, as well as the phenotype of the hybrid, influence the breeder'sdecision whether to continue with the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes into parentlines. These lines are used to produce new cultivars. New cultivars canbe evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. An F₂ population is produced byselfing one or several F₁′s. Selection of the best individuals in thebest families is performed. Replicated testing of families can begin inthe F₄ generation to improve the effectiveness of selection for traitswith low heritability. At an advanced stage of inbreeding (i.e., F₆ andF₇), the best lines or mixtures of phenotypically similar lines aretested for potential release as new cultivars.

Backcross breeding and cross breeding have been used to transfer genesfor a simply inherited, highly heritable trait into a desirablehomozygous cultivar or inbred line, which is the recurrent parent. Thesource of the trait to be transferred is called the donor parent. Theresulting plant obtained from a successful backcrossing program isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. After multiple backcrossing generations with selection, theresulting line is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Cross breeding or backcross breeding of a DM resistant cucumber plantmay be conducted where the other parent (second cucumber plant) is DMresistant or the other parent is not DM resistant.

Cucumber plants generated of the invention may be generated using asingle-seed descent procedure. The single-seed descent procedure, in thestrict sense, refers to planting a segregating population, thenselecting one plant in this and each subsequent generation to self andcreate the next generation. When the population has been advanced fromthe F₂ to the desired level of inbreeding, the plants from which linesare derived will each trace to different F₂ individuals. The number ofplants in a population declines each generation due to failure of someseeds to germinate or some plants to produce at least one seed. As aresult, not all of the F₂ plants originally sampled in the populationwill be represented by a progeny when generation advance is completed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several availablereference books (e.g., Fehr, Principles of Cultivar Development Vol. 1,pp. 2-3 (1987)).

In one aspect of the present invention, the source of DM resistancetrait for use in a breeding program is derived from a plant selectedfrom the group consisting of PI197088, ASL147-2027, EUR154-1012GY,EUR154-1021GY, GSP33-1094GY, GPN33-1093GY, 03/8020-20_TUP03_DMFL_(—)1,03/8024-19_TUP03_DMFL_(—)1, and 03/8039-5_TUP03_DMFL_(—)1, and DMresistant progeny thereof, as described in U.S. Patent ApplicationPublication 2009-0265083. In another aspect, the source of the DMresistance trait for use in a breeding program is not derived from aplant selected from the group consisting of PI197088, ASL147-2027,EUR154-1012GY, EUR154-1021GY, GSP33-1094GY, GPN33-1093GY,03/8020-20_TUP03_DMFL_(—)1, 03/8024-19_TUP03_DMFL_(—)1, and03/8039-5_TUP03_DMFL_(—)1, and DM resistant progeny thereof.

Another aspect of the invention is directed to an inbred cucumber planthaving resistance to DM, wherein said resistance is exhibited when saidplant is in contact with said DM, and wherein said cucumber plant is notderived from a plant selected from the group consisting of ASL147-2027,EUR154-1012GY, EUR154-1021GY, GSP33-1094GY, GPN33-1093GY,03/8020-20_TUP03_DMFL_(—)1, 03/8024-19_TUP03_DMFL_(—)1, and03/8039-5_TUP03_DMFL_(—)1. Also included in the invention is a cucumberplant having a genome, wherein said genome comprises a genetic locusconferring resistance to DM, wherein said genetic locus contains one ormore genetic markers linked to said genetic locus conferring resistanceto DM, and wherein said cucumber plant is not accession PI197088.

In another aspect, additional sources of DM resistance for use in abreeding program can be identified by screening cucumber germplasm forresistance to DM. In yet another aspect, cucumber plants can be screenedfor DM resistance by identifying germplasm exhibiting reduced diseasesymptoms relative to a control cucumber plant after inoculation orinfection. In one aspect, cucumber plants can be screened for resistanceto DM using a test such as a field or greenhouse screen and diseaserating schemes as described in Example 1, Example 2, or Example 8.

In another aspect, additional sources of DM resistance for use in abreeding program can be identified by screening with one or moremolecular markers linked to a genetic locus conferring resistance to DM,such as those identified herein.

In another aspect, additional sources of DM resistance for use in abreeding program can be identified by a combination of screeningcucumber plants for reduced disease symptoms then screening with one ormore molecular markers linked to a genetic locus contributing toresistance to DM.

In another aspect, cucumber lines having DM resistance can be used inbreeding programs to combine DM resistance with additional traits ofinterest. In one aspect, DM resistance can be combined with anyadditional trait, including disease resistant traits, yield traits, andfruit quality traits. For example, breeding programs can be used tocombine the DM resistance trait with alleles that contribute to size andshape in cucumber fruit. Breeding programs can also be used to combineDM resistance with one or more disease resistant traits. Such diseaseresistant traits include, without limitation, resistance to:Verticillium wilt, root knot nematodes, Tobacco Mosaic Virus, Cucumberscab, Powdery mildew, Target spot, Cucumber Mosaic Virus, and Fusariumwilt. In another aspect, the traits that are combined can beco-inherited in subsequent crosses.

The present invention also provides for parts of the DM resistantcucumber plants produced by a method of the present invention. Parts ofcucumber plants, without limitation, include plant cells or parts ofplant cells, seed, endosperm, meristem, flower, anther, ovule, pollen,fruit, flowers, stems, roots, stalks or leaves, scions, and root stocks.Plant parts also include the parts of a cucumber fruit, which includethe placenta, columella and pericarp. In one embodiment of the presentinvention, the plant part is a seed.

The invention further provides for parts of a cucumber plant having agenome, that comprises at least one genetic locus giving rise to DMresistance in the cucumber plant. In another embodiment, parts ofcucumber plants are derived from a cucumber plant selected from thegroup consisting of the deposited cucumber lines described in U.S.Patent Application Publication 2009-0265083, and DM resistant progenythereof. In accordance with one aspect of the present invention, thephysiological and morphological characteristics of deposited linesASL147-2027, EUR154-1012GY, EUR154-1021GY, GSP33-1094GY, GPN33-1093GY,03/8020-20_TUP03_DMFL_(—)1, 03/8024-19_TUP03_DMFL_(—)1, and03/8039-5_TUP03_DMFL_(—)1 are set forth in Tables 1-8 below.

TABLE 1 Physiological and Morphological Characteristics of LineASL147-2027 Mo. CHARACTERISTIC ASL147-2027 1. Type Cucumber PredominateUsage Slicing Predominate Culture Outdoor Area of Best Adaptation in USAMost Areas 2. Maturity Days from Seeding to Market 50-55 3. Plant HabitVine Growth Indeterminate Sex Monoecious Flower Color Yellow 4. Fruit atEdible Maturity Fruit Neck Shape Not Necked Fruit Tapering Ends Blunt orRounded Skin Thickness Thick Skin Ribs Ribbed Skin Toughness Tough SkinLuster Dull Spine Color White Spine Quality Coarse Spine Density FewFlavor Bitterfree 5. Insect Resistance Aphid (Aphis gossypii)Susceptible

TABLE 2 Physiological and Morphological Characteristics of LineEUR154-1012 GY. CHARACTERISTIC EUR 154-1012 GY 1. Type CucumberPredominate Usage Fresh Predominate Culture Greenhouse Area of BestAdaptation in USA Spain 2. Maturity Days from Seeding to Market 60-65 3.Plant Habit Vine Growth Indeterminate Sex Gynoecious Flower Color Yellow4. Stem Length 150-200 cm Internode Length   5-8 cm Stem FormGrooved-Ridged 5. Fruit at Edible Maturity Length  30-32 cm Diameter atMedial  45-50 mm Weight 300-400 gm Skin Color Medium green YellowishBlossom End Strips No Predominant Color at Stem End Uniform greenPredominant Color at Blossom End Uniform green Fruit Neck Shape MediumFruit Tapering Rounded Stem End Cross Section rd Medial Cross Section rdBlossom End Cross Section rd Skin Thickness Thin Skin Ribs Ribbed SkinToughness Low Skin Luster Shiny Spine Color White Spine Quality FineSpine Density Very low Tubercles (Warts) No Flavor Bitterfree 6. Fruitat Harvest Maturity Length  35-37 cm Diameter at Medial  50-60 mm ColorYellow Color Pattern Striped Surface Smooth Netting Slight or none FruitSet Normally without seeds 7. Seeds No. per Fruit 30-80 Per 1,000 Seeds 30-35 gm 8. Disease Resistance Cucumber Scab (Gumosis) (Cladosporiumcucumerinum) Downy Mildew Resistant Powdery Mildew (Erysiphe Resistantcichoracearum) Cucumber Mosaic Virus Susceptible Cucumber Vein YellowingVirus Susceptible Cucumber yellow Stunted Disorder Intermediateresistance Virus 9. Insect Resistance Aphid (Aphis gossypii) Susceptible

TABLE 3 Physiological and Morphological Characteristics of Line EUR154-1021 GY. CHARACTERISTIC EUR 154-1021 GY 1. Type Cucumber PredominateUsage Fresh Predominate Culture Greenhouse Area of Best Adaptation inUSA Spain 2. Maturity Days from Seeding to Market 60-65 3. Plant HabitVine Growth Indeterminate Sex Gynoecious Flower Color Yellow 4. StemLength 150-200 cm Internode Length  50-80 mm Stem Form Grooved-Ridged 5.Fruit at Edible Maturity Length  30-32 cm Diameter at Medial  45-50 mmWeight 300-400 gm Skin Color Medium green Yellowish Blossom End StripsNo Predominant Color at Stem End Uniform green Predominant Color atBlossom End Uniform green Fruit Neck Shape Not necked Fruit TaperingRounded Stem End Cross Section rd Medial Cross Section rd Blossom EndCross Section rd Skin Thickness Thin Skin Ribs Ribbed Skin Toughness LowSkin Luster Shiny Spine Color White Spine Quality Fine Spine Density LowTubercles (Warts) No Flavor Bitterfree 6. Fruit at Harvest MaturityLength  35-37 cm Diameter at Medial  50-60 mm Color Yellow Color PatternStriped Surface Smooth Netting Slight Fruit Set Normally without seeds7. Seeds No. per Fruit 30-80 Per 1,000 Seeds  30-35 gm 8. DiseaseResistance Downy Mildew Resistant Powdery Mildew (Erysiphe Resistantcichoracearum) Cucumber Mosaic Virus Susceptible Cucumber Vein YellowingVirus Susceptible Cucumber yellow Stunted Disorder Intermediateresistance Virus 9. Insect Resistance Aphid (Aphis gossypii) Susceptible

TABLE 4 Physiological and Morphological Characteristics of Line GSP33-1094 GY. CHARACTERISTIC GSP 33-1094 GY 1. Type Cucumber PredominateUsage Pickling Predominate Culture Outdoor Area of Best Adaptation inUSA Most Areas 2. Maturity Days from Seeding to Market  60-62 3. PlantHabit Vine Growth Indeterminate Sex 100% Gynoecious Flower Color Yellow4. Stem Length 150-200 cm Number of Nodes from Cotyledon  3-4 Leaves toNode Bearing the First Pistillate Flower Internode Length  20-30 StemForm Grooved-Ridged 5. Leaf Mature Blade of Third Leaf Length 200-250 mmWidth 150-200 mm Petiole Length  6.5-8 6. Fruit at Edible MaturityLength  12-14 cm Diameter at Medial  35-45 Weight  80-120 gm Skin ColorMottled or Speckled with yellow Yellowish Blossom End Strips Extend Lessthan ⅓ of the Fruit Length Predominant Color at Stem End Medium GreenPredominant Color at Blossom Light Green End (Arlington White Spine)Fruit Neck Shape Not Necked Fruit Tapering Ends Blunt or Rounded StemEnd Cross Section Square Medial Cross Section Square Blossom End CrossSection Square Skin Thickness Thick Skin Ribs Ribbed Skin ToughnessTender Skin Luster Dull Spine Color White Spine Quality Fine SpineDensity Many Tubercles (Warts) Few, Prominent (Salad) Flavor Bitterfree7. Fruit at Harvest Maturity Length  25-30 cm Diameter at Medial  10-13Color Cream Color Pattern Not Striped Surface Smooth Netting Slight orNone Fruit Set Parthenocarpically 8. Seeds No. per Fruit 150-200 Per1,000 Seeds  22-25 gm 9. Disease Resistance Cucumber Scab (Gumosis)Susceptible (Cladosporium cucumerinum) Downy Mildew Resistant PowderyMildew (Erysiphe Resistant cichoracearum) Target Spot (CorynesporaSusceptible cassiicola) Cucumber Mosaic Virus Resistant 10. InsectResistance Aphid (Aphis gossypii) Susceptible

TABLE 5 Physiological and Morphological Characteristics of Line GPN33-1093 GY. CHARACTERISTIC GPN 33-1093 GY 1. Type Cucumber PredominateUsage Pickling Predominate Culture Outdoor Area of Best Adaptation inUSA Most Areas 2. Maturity Days from Seeding to Market  60-65 3. PlantHabit Vine Growth Indeterminate Sex Primarily Gynoecious Flower ColorYellow 4. Stem Length 150-200 cm Number of Nodes from Cotyledon  2-3Leaves to Node Bearing the First Pistillate Flower Internode Length 20-25 Stem Form Grooved-Ridged 5. Leaf Mature Blade of Third LeafLength 200-230 mm Width 150-200 mm Petiole Length  4-8 6. Fruit atEdible Maturity Length  11-13 cm Diameter at Medial  35-45 Weight 80-120 gm Skin Color Mottled or Speckled with yellow Yellowish BlossomEnd Strips Extend Less than ⅓ of the Fruit Length Predominant Color atStem End Medium Green Predominant Color at Blossom Light Green End(Arlington White Spine) Fruit Neck Shape Not Necked Fruit Tapering EndsBlunt or Rounded Stem End Cross Section Square Medial Cross SectionSquare Blossom End Cross Section Square Skin Thickness Thick Skin RibsRibbed Skin Toughness Tender Skin Luster Dull Spine Color White SpineQuality Fine Spine Density Few Tubercles (Warts) Few, Prominent (Salad)Flavor Bitterfree 7. Fruit at Harvest Maturity Length  25-30 cm Diameterat Medial  10-13 Color Cream Color Pattern Striped Surface SmoothNetting Slight or None Fruit Set Normally with Seeds 8. Seeds No. perFruit 150-200 Per 1,000 Seeds  22-25 gm 9. Disease Resistance CucumberScab (Gumosis) Susceptible (Cladosporium cucumerinum) Downy MildewResistant Powdery Mildew (Erysiphe Resistant cichoracearum) Target Spot(Corynespora Susceptible cassiicola) Cucumber Mosaic Virus Resistant 10.Insect Resistance Aphid (Aphis gossypii) Susceptible

TABLE 6 Physiological and Morphological Characteristics: Line03/8020-20_TUP03_DMFL_1. CHARACTERISTIC 03/8020-20_TUP03_DMFL_1 1. TypeCucumber Predominate Usage Fresh Predominate Culture Outdoor Area ofBest Adaptation in USA Turkey & ½ East 2. Maturity Days from Seeding toMarket  60-65 3. Plant Habit Vine Growth Indeterminate Sex MonoeciousFlower Color Yellow 4. Stem Length 150-200 cm Internode Length  30-50 mmStem Form Grooved-Ridged 5. Fruit at Edible Maturity Length  12-15 cmDiameter at Medial  35-45 Weight  80-120 gm Skin Color Green YellowishBlossom End Strips No Predominant Color at Stem End Uniform greenPredominant Color at Blossom End Uniform green Fruit Neck Shape NotNecked Fruit Tapering Ends Blunt or Rounded Stem End Cross Section rdMedial Cross Section rd Blossom End Cross Section rd Skin Thickness ThinSkin Ribs Not ribbed Skin Toughness Low Skin Luster Shiny Spine ColorWhite Spine Quality Fine Spine Density High Tubercles (Warts) No FlavorBitterfree 6. Fruit at Harvest Maturity Length  18-20 cm Diameter atMedial  30-45 mm Color Cream Color Pattern Striped Surface SmoothNetting Slight or None Fruit Set Normally with Seeds 7. Seeds No. perFruit 150-200 Per 1,000 Seeds  22-25 gm 8. Disease Resistance CucumberScab (Gumosis) Resistant (Cladosporium cucumerinum) Downy MildewResistant (Pseudoperonospora cubensis) Powdery Mildew (ErysipheSusceptible cichoracearum) Cucumber Mosaic Virus Susceptible 9. InsectResistance Aphid (Aphis gossypii) Susceptible

TABLE 7 Physiological and Morphological Characteristics: Line03/8024-19_TUP03_DMFL_1. CHARACTERISTIC 03/8024-19_TUP03_DMFL_1 1. TypeCucumber Predominate Usage Fresh Predominate Culture Outdoor Area ofBest Adaptation in USA Turkey & ½ East 2. Maturity Days from Seeding toMarket  60-65 3. Plant Habit Vine Growth Indeterminate Sex MonoeciousFlower Color Yellow 4. Stem Length 150-200 cm Internode Length  30-50 mmStem Form Grooved-Ridged 5. Fruit at Edible Maturity Length  15-17 cmDiameter at Medial  35-45 Weight  80-120 gm Skin Color Green YellowishBlossom End Strips No Predominant Color at Stem End Uniform greenPredominant Color at Blossom End Uniform green Fruit Neck Shape Notnecked Fruit Tapering Ends blunt or rounded Stem End Cross Section rdMedial Cross Section rd Blossom End Cross Section rd Skin Thickness ThinSkin Ribs Not ribbed Skin Toughness Low Skin Luster Shiny Spine ColorWhite Spine Quality Fine Spine Density Low Tubercles (Warts) No 6. Fruitat Harvest Maturity Length  12-23 cm Diameter at Medial  30-45 mm ColorCream Color Pattern Striped Surface Smooth Netting Slight or none FruitSet Normally with seeds 7. Seeds No. per Fruit 150-200 Per 1,000 Seeds 22-25 gm 8. Disease Resistance Cucumber Scab (Gumosis) Susceptible(Cladosporium cucumerinum) Downy Mildew Resistant Powdery Mildew(Erysiphe Intermediate resistance cichoracearum) Cucumber Mosaic VirusResistant 9. Insect Resistance Aphid (Aphis gossypii) Susceptible

TABLE 8 Physiological and Morphological Characteristics: Line03/8039-5_TUP03_DMFL_1. CHARACTERISTIC 03/8039-5_TUP03_DMFL_1 1. TypeCucumber Predominate Usage Fresh Predominate Culture Outdoor Area ofBest Adaptation in USA Turkey & ½ East 2. Maturity Days from Seeding toMarket  60-65 3. Plant Habit Vine Growth Indeterminate Sex MonoeciousFlower Color Yellow 4. Stem Length 150-200 cm Internode Length  30-50 mmStem Form Grooved-Ridged 5. Fruit at Edible Maturity Length  16-18 cmDiameter at Medial  35-45 Weight  80-120 gm Skin Color Green YellowishBlossom End Strips No Predominant Color at Stem End Uniform greenPredominant Color at Blossom End Uniform green Fruit Neck Shape Notnecked Fruit Tapering Ends blunt or rounded Stem End Cross Section rdMedial Cross Section rd Blossom End Cross Section rd Skin Thickness ThinSkin Ribs Not ribbed Skin Toughness Low Skin Luster Shiny Spine ColorWhite Spine Quality Fine Spine Density Medium Tubercles (Warts) NoFlavor Bitterfree 6. Fruit at Harvest Maturity Length  20-24 cm Diameterat Medial  30-45 mm Color Cream Color Pattern Striped Surface SmoothNetting Slight or None Fruit Set Normally with Seeds 7. Seeds No. perFruit 150-200 Per 1,000 Seeds  22-25 gm 8. Disease Resistance CucumberScab (Gumosis) Resistant (Cladosporium cucumerinum) Downy MildewResistant Powdery Mildew (Erysiphe Intermediate resistancecichoracearum) Cucumber Mosaic Virus Susceptible 9. Insect ResistanceAphid (Aphis gossypii) Susceptible

In one embodiment, the invention provides a DM resistant cucumber plant,or the fruit or seeds thereof, wherein the cucumber plant demonstrates areduction in foliar symptoms of chlorotic and/or necrotic lesionsrelative to a non-resistant control plant upon inoculation or infectionwith DM, and wherein said plant demonstrates resistance to one or moreof Verticillium wilt, root knot nematodes, tobacco mosaic virus,cucumber scab, powdery mildew, target spot, cucumber mosaic virus,papaya ringspot virus, zucchini yellow mosaic virus, and Fusarium wilt.In another embodiment, those cucumber plants, or the fruit or seedsthereof, are selected from DM resistant progeny of lines described inTables 1-8. In other embodiments, a DM resistant cucumber plant thatalso demonstrates resistance to one or more of: Verticillium wilt,cucumber scab, powdery mildew, target spot, cucumber mosaic virus,nematodes, tobacco mosaic virus papaya ringspot virus, zucchini yellowmosaic virus, and Fusarium wilt displays a greater than 10% reduction,or a greater than 30% reduction, or a greater than 60% reduction infoliar symptoms of chlorotic and/or necrotic lesions upon inoculation orinfection with DM. In some aspects, the cucumber plants are adaptedeither for greenhouse growth or for field growth.

One aspect of the invention provides a DM cucumber plant, or the fruitor seeds thereof, wherein the cucumber plant, or the fruit thereof,expresses one, or two, or three, or more independently selecteddesirable traits in addition to DM resistance. In one embodiment, the“desirable trait” or “desirable traits” are selected from the groupconsisting of: fruit size, shape, color, surface appearance; seednumber, seed size, locule number; pericarp thickness and toughness;taste, bitterness, the presence of tubercles, and shelf life, plantvigor, leaf shape, leaf length, leaf color, plant height, whether theplant is determinate or not, time to maturity, adaptation to fieldgrowth, adaptation to greenhouse growth, and resistance to one or morediseases or disease causing organisms such as Verticillium Wilt, rootknot nematodes, Tobacco Mosaic Virus, Cucumber Scab, Powdery Mildew,Downy Mildew, Target Spot, Cucumber Mosaic Virus, and Fusarium Wilt. Inanother embodiment the “desirable trait” or “desirable traits” areselected from the group consisting of: fruit size, fruit shape, fruitcolor, fruit taste, the number of seeds per fruit, the size of seeds,the thickness of fruit pericarp tissue, the shelf life of fruit,resistance to Verticillium Wilt, resistance to Cucumber Scab, resistanceto Powdery Mildew, resistance to Target Spot, resistance to CucumberMosaic Virus, resistance to nematodes, resistance to Tobacco MosaicVirus, resistance to Papaya Ringspot Virus, resistance to ZucchiniYellow Mosaic virus, and resistance to Fusarium Wilt. In yet anotherembodiment, the “desirable trait” or “desirable traits” are selectedfrom the group consisting of: fruit size, fruit shape, fruit color,fruit taste, the shelf life of fruit, resistance to Cucumber scab,resistance to Powdery mildew, resistance to Target spot, and resistanceto Cucumber mosaic Virus. In still another embodiment the “desirabletrait” or “desirable traits” are selected from the group consisting of:fruit size, fruit shape, fruit color, fruit quality acceptable tomarket, and the shelf life of fruit.

In other aspects of the invention, the plants bearing one or moredesirable traits in addition to DM resistance display a greater than10%, or a greater than 30%, or a greater than 60%, or a greater than 80%reduction in foliar symptoms of chlorotic and/or necrotic lesionsrelative to a non-resistant control plant upon inoculation or infectionwith DM. Another aspect of the present invention is directed to a methodof producing a DM resistant cucumber plant comprising: crossing acucumber line having DM resistance with a second plant lacking DMresistance but capable of donating one or more of the aforementioneddesirable traits.

EXAMPLES Example 1 Downy Mildew Culture and Disease Screening—Field

Pseudoperonospora cubensis (Berk. et Curt.) Rostow is an obligatepathogen. Therefore, it must be maintained on live plants of asusceptible cucurbit. Two isolates were used in screening for resistancein this study. The “old” isolate of P. cubensis is characterized by itspathogenicity on both squash and cucumber. The “new” isolate of P.cubensis is not considered pathogenic on squash but is very pathogenicon cucumber. The pathogen was stored by freezing leaves or cotyledonswith abundant sporulation at −80° C. Although there may be some loss inspore viability inherent in the freezing process, no decrease inviability over time once spores are frozen, has been found. Six weeksbefore spreader host plants were to be transplanted in the field,susceptible cucumber hosts were planted in a controlled environmentchamber. At three weeks they were inoculated with a spore suspensionderived from infected leaves stored in a −80° C. freezer. Inoculatedculture plants were maintained at 20° C.; once chlorotic lesions havedeveloped, plants were placed in the dew chamber overnight to inducesporulation. This culture was transferred weekly on cucumber untilplants were transplanted into the field.

Trials were direct seeded in the field. Spreader rows of susceptiblecucumber were planted in every third row. When spreader rows weretwo-three weeks old, infected plants (reared in the growth room) weretransplanted within the spreader rows. Breeder trials were donesimultaneously. Plots were maintained in good horticultural condition,consistent with techniques normally employed for culture of cucumbers inthe Southeastern United States.

Spreader plants at the three to four leaf stage were inoculated in thegreenhouse by misting with sporangial suspension using a spray bottle.Inoculum was formulated in sterile distilled water. After inoculation,plants were placed in a dew chamber at 100% RH and 20° C., for 18-24hours. Spreader plants were transplanted to the field where a solid setsprinkler provided a nightly moist period to encourage development andspread of disease.

Tests were evaluated once symptoms had developed on the susceptiblecheck, sometimes called the susceptible control. Controls includingPI197088 (resistant control); DMP21, GP14, LLP 1, POINSETT 76,(intermediate-resistant controls); and SPRINT440, MARAM, and SMR58(susceptible controls) were used. Three observations were made on eachplot, one at each end and one in the middle. The mean disease index foreach plot was calculated. These were averaged for all three replicatesand the standard deviation was determined. The disease index ranges forthe categories “Resistant,” “Intermediate Resistant” and “Susceptible”were then determined. Varieties were generally trialed several timesbefore a final disease resistance level determination was made. Arandomized complete block design was used in the disease test. Each linewas replicated three times—approximately 40 plants per entry weretested. Lines with limited seed available were included as a single repobservation plot. Checks were included as entries to gauge the severityof the test. Plots were 12 feet long with a 3-foot alley between ends ofblocks. A susceptible spreader was planted in every third row and on theoutside borders of the entire planting

Example 2 Downy Mildew Culture and Disease Screening—Greenhouse

Pseudoperonospora cubensis (Berk. et Curt.) Rostow, as described andstored above in Example 1, was also used for greenhouse screens. Twoweeks prior to screen inoculation, susceptible cucumber hosts were sownin seedling trays. At one week post planting, the seedlings areinoculated with a rate of approximately 5×10⁴ sporangia/ml. Theinoculated hosts were then placed into a growth chamber and maintainedfor seven days at about 70° F. After seven days, the seedlings wereplaced into a dew chamber overnight to induce sporulation. This culturewas transferred onto susceptible cucumber hosts on a weekly basis.

Cotyledon screens were planted in seedling trays. Susceptible andresistant checks were planted on both sides of each tray. Plants wereseeded and maintained in a greenhouse at 80° F. Inoculation wasconducted at 7 to 10 days for cotyledons and at the 5th leaf stage fortrue leaves. Plants were inoculated by misting with sporangialsuspension using a spray bottle at a concentration of about 5×10⁴sporangia per ml for cotyledons and 1×10⁴ to 3×10⁴ for true leaves.After inoculation, plants were placed in a dew chamber at 100% relativehumidity and 20° C., for 18-24 hours.

Tests were evaluated once symptoms have developed on the susceptiblecheck, sometimes called the susceptible control. Controls used werePI197088 (resistant control) MARAM (susceptible control), and SMR58(resistant control). Resistant and intermediate survivors of thecotyledon screens were kept and transplanted into 3-inch peat pots to beinoculated again or to be transplanted into greenhouse grow bags.Resistant and intermediate survivors of the true leaf screens weretransplanted directly into greenhouse grow bags.

Example 3 Introgression of DM resistance into Cucumber Lines

Downy Mildew resistance identified in the Plant Introduction linePI197088 was found to be stable in multiple screening locationsworldwide and against both older (pathogenic on squash and cucumber) andnewly emerged (not considered pathogenic on squash and very pathogenicon cucumber) isolates of P. cubensis. However, both plants and fruit ofPI197088 are commercially unacceptable. A locus contributing to DownyMildew resistance in PI197088 was mapped with molecular markers asdescribed in Example 4. A total of about 128 cucumber lines wereseparately screened using one or both DM isolates. DNA was isolated fromresistant lines to screen for marker polymorphisms between the donor andrecurrent parents.

Included in these screens were cucumber varieties Conquistador,Crispina, DMP21, and PI197088, among others, which are resistant orintermediate-resistant. Also included were Colt, Sprint440, Talladega,Lucinde, and Serena, among others, as susceptible control lines. Tissuesamples from each of the DM resistant lines were collected for use inDNA analysis and production of a DNA library to identify markersassociated with DM resistance. Seeds were also obtained from each of thelines demonstrating DM resistance generally via mixed pollenpollinations within each accession, and where possible via selfing.Mixed pollen pollinations were generally used in wild type cucumbers asthey often contain a self-incompatibility factor.

Initial crosses were made between PI197088 and a recurrent susceptibleparent (cv. Lucinde) to create F₁ plants. Plants derived from thesecrosses were used for disease testing as described in Examples 1 and/or2. Experiments were performed to screen for DM resistance on acollection of elite lines that show horticulturally acceptable plant andfruit types, and should have the DM resistance introgressed fromPI197088. These tests were performed in three locations (Woodland,Calif., Tifton, Ga., and Wageningen, NL) and used two isolates ofPseudoperonospora cubensis: an “older” isolate, pathogenic on squash andcucumber, and the putative “new” isolate, not considered pathogenic onsquash but very virulent on cucumber. Simultaneously, these samples weregenotyped with molecular markers to identify a QTL contributing DMresistance in PI197088 (see also Example 4). These tests associate theDM pathology response with the presence of an allele from PI197088.During this time, breeders submitting the samples assembled all trialdata available on these lines in which plant and fruit types are notedor quantified. The following lines shown in Tables 9-10 were screenedfor resistance to Downy Mildew.

TABLE 9 Pedigrees for Cucumber Lines listed in U.S. Patent ApplicationPublication 2009-0265083, for which a Seed Deposit was Made. CucumberLine Pedigree ASL147-2027 PI-197088-MO/ASL-1105-GY:@.1.1.1.1.4. EUR154-[(ALCOR(WMV) × VENTURA × PIDM/NIZ335 * 2) × (ALCOR(V) × VENTURA × 1012GYPIDM/CARMEN * 2)] × [(ALCOR(V) × VENTURA × PIDM/ CARMEN * 2) × (ALCOR(V)× VENTURA × PIDM/CARMEN * 2)] EUR154- (ALCOR(WMV) × VENTURA ×PIDM/NIZ335 * 2) × (ALCOR(V) × VENTURA × 1021GY PIDM/CARMEN * 2) GSP33-F9-(Jazz/5/Sal//SMR-58Nim/PiHoNi/3/NO-50/4/H-171wit/SMR- 1094GY58Nim//Carol/3/NO-50 * Harmonie) GPN33-F8-(Jazz/5/Sal//SMR-58Nim/PiHoNi/3/NO-50/4/H-171wit/SMR- 1093GY58Nim//Carol/3/NO-50) 03/8020- BA.KO {(147W * PI) * 225)} * (BA MO *part) BC403/ 20_TUP03_DMFL_1 8020-20_TUP03_DMFL_1 + ---1_TUNE03_DM-2_TUp05_TKFA06 03/8024- BA.KO {(147W * PI) * 225)} * 19_TUP03_DMFL_1[(me/n * 147wmv)bc4f5 * (bamo * parth)bc4f5] 03/8024-19_TUP03_DMFL_1 +---4_TUNE03_DM- 1_TUp05_TKFA06 03/8039- BA.KO {(147W * PI) * 225)} * HP159] * [(BA MO * part) BC403/ 5_TUP03_DMFL_1 8039-5_TUP03_DMFL_1 +---3_TUNE03_DM- 4_TUp05_TKFA06

TABLE 10 Marker haplotypes and associated Downy Mildew (DM) reactionscores for five markers in the DM resistance QTL region. cM¹ posi- Hap².Marker tion 1 Hap. 2 Hap. 3 Hap. 4 Hap. 5 Markers and sample haplotypesCAPs_ENK60 4  SUS³ SUS  RES⁴ RES RES CAPs_ENK59 5 SUS SUS RES RES RESCAPs_17170 11 SUS SUS SUS SUS RES CAPs_17179 11 SUS SUS SUS SUS RESCAPs_17563/66 39 SUS RES SUS RES RES Downy Mildew summary statisticsassociated with marker haplotypes Mean (DM⁵) 4.8 4.5 4.0 2.2 3.3 Minimum(DM) 4.3 3.3 3.7 1.0 1.0 Maximum (DM) 5.0 5.0 4.3 3.3 5.0 Std. 0.2 0.60.3 0.5 1.2 Deviation (DM) Number of 3 3 1 19 11 lines with haplotypeData represent thirty seven cucumber lines. ¹cM = centiMorgans. ²Hap =Haplotype at five markers in DM QTL. ³SUS = Downy Mildew susceptibleallele associated with Lucinde parent in mapping population. ⁴RES =Downy Mildew resistance allele associated with PI197088 parent inmapping population. ⁵DM = phenotypic scores in a pathology test forDowny Mildew.

Thirty seven cucumber lines were tested for reactions toPseudoperonospora cubensis in a controlled pathology screen. Eighteenplants were tested in three replicates of six plants. From eachreplication, three plants (total from three replications=9) weregenotyped for five markers defining the DM QTL region. From these data,consensus marker genotypes and DM summary statistics were developed foreach line. These data are summarized in Table 10. The five markers usedin this test were selected from the eight linked markers in the DM QTL.The five markers were selected based on reliable performance in thelaboratory, and/or associations with DM phenotype that were moreconsistent than the other markers.

Table 10 supports association of the haplotype RES-RES-SUS-SUS-RES atthe markers CAPs_ENK60, CAPs_ENK59, CAPs_(—)17170, CAPs_(—)17179,CAPs_(—)17563/66 with a more DM resistant phenotype. Substitution of theSUS allele with the RES allele at markers CAPs_ENK60, CAPs_ENK59,CAPs_(—)17563/66 yields a change in mean DM phenotype from 4.8 to 2.2 intests where the rating scale is 1=resistant and 5=susceptible.

Example 4 Marker Analysis of DM Resistant Cucumber Plants

Resistant plants are analyzed using genetic markers distributedthroughout the cucumber genome. Genetic markers for Cucumis areavailable from a variety of sources such as USDA-ARS (Vegetable CropsResearch Unit—Department of Horticulture, University ofWisconsin-Madison). A larger set of markers was pre-screened on theparental lines and polymorphic markers were selected, from among thepre-screened markers, for a subsequent screen. A correlation was thenestablished with most of the resistant plants and the presence ofspecific donor alleles, for instance as shown in Table 10 and FIG. 2.Most of the resistant plants contained introgressed DNA from theresistant donor line, PI19788, for instance at loci as shown in FIG. 2.A multiple regression model was constructed to retain the markers thatcontributed to the DM resistance phenotype. In this analysis, markersCAPs_ENK60, CAPs_(—)17170, and CAPs_(—)17563/66 remained significant,generating a model with R² of 0.47.

Primer pairs and reaction conditions utilized to identify alleles atgiven markers on chromosome 5, to define the QTL for DM resistance inCucumis sp., are shown in Table 11 and Table 12. For marker 17179, thesame reverse primer was used for the two alleles.

TABLE 11 Primer pairs used (SEQ ID Nos: 1 to 19)to identify alleles of QTL on chromosome 5. Forward PrimerReverse Primer Electrophoresis Marker name (5′ to 3′) (5′ to 3′) Enzymecondition Notes CAPs_21826 TCAAGCCATAGTCTA CGCTATATCATGGATGGC NsiI 3%ACCCATGC TAGAAAT agarose gel CAPs_ENK60 GAATAGATAGGCTACGTATAAAACTTGAGTGAA HpyC 3% ACTTTTCCCTCTTG TTTAATGCATGAA H4 IVagarose gel CAPs_ENK59 TGTTTCATAACTACA TAGTTTCTTTCTTGCTGG 3%GCTTCATGTTAAATA ACGAACC agarose gel TTACT CAPs_17170 TATGGGCTATGTGAAAGCGTGACAACTACAAAA Aft III 3% ACTCTT CAT agarose gel CAPs_17179GAAATAAATGGATGA GTTCGTTGATCAGTGTGA Capillary Forward AGCGAGGA TATTTCAATprimer for PI197088 allele CAPs_17179 ATCGGTCTTTGCCAC GTTCGTTGATCAGTGTGACapillary Forward CTTTTG TATTTCAAT primer for Lucinde allele CAPs_18229TGTTTGGAAGGGTTT TGCCATGTCGCCAACAGT HindIII 3% CTTGGG agarose gelCAPs_17563/66 AGGAGGGACAGAGA TCCGTTTTAGGTGATTGT Capillary GAATTTGATATAATCAAATACAT CAPs_ENK70 AAAGTTGATAGTGCA TCCGCTTATGGGTTTTTG Taq1 3%TGAGTTGGTAAAATA TGAG agarose gel UBC12-1200 TATGGGCTATGTGAAAGCGTGACAACTACAAAA ACTCTT CAT

TABLE 12 Reaction conditions for PCR. Master Component Combine per 10 ulrxn Mix for 10: PCR for: Markers run on agarose gel (CAPs_ENK60,CAPs_ENK59, CAPs_17170, CAPs_18229, CAPs_21826, CAPs_ENK70 HotStart-ITTaq Master Mix (2X) 5 50 5 uM Forward Primer 0.53 5.3 5 uM ReversePrimer 0.53 5.3 MQ H₂O 3.14 31.4 Template DNA 0.8 8 Sum 10 100 PCR for:CAPs_17179 HotStart-IT Taq Master Mix (2X) 5 50 5 uM Primer 1 0.53 5.3 5uM Primer 2 0.53 5.3 5 uM Tailed Primer 0.053 0.53 Labeled primer 14750.53 5.3 MQ H₂O 2.557 25.57 Template DNA 0.8 8 Sum 10 100 PCR for:CAPs_17563/66 HotStart-IT Taq Master Mix (2X) 5 50 5 uM Primer 0.53 5.35 uM Tailed Primer 0.053 0.53 Labeled primer 1475 0.53 5.3 MQ H₂O 3.08730.87 Template DNA 0.8 8 Sum 10 100

Analysis for the genetic markers of Table 11 was performed by PCRamplification. PCR reactions were conducted as follows: PCR reactionscontain 1.0 microliters of cucumber genomic DNA (10 ng), 2 μl 10× PCRBuffer (ABI PCR Buffer I: part no. N808-0006), 1.0 μl 10× dNTP mix(final concentration of each dNTP is 250 μM), 1 μl each primer (5picomoles of each primer), 0.2 μl Taq Polymerase (1 unit), and sterilewater to a total volume of 20 microliters. PCR reactions are incubatedfor 2 minutes at 94° C., 30 seconds at 94° C., 30 seconds at 50° C., and90 seconds at 72° C. for 35 cycles, followed by a single cycle of 72° C.for 5 minutes. PCR reactions are performed for instance on an ABI9700PCR machine (Applied Biosystems, Foster City, Calif.).

Sequencing of genomic DNA flanking the loci initially screened in theQTL region could explain some of the variability that was seen in someDM resistance scores. For instance, in certain lines with resistanthaplotypes, but variable resistance scores, variability could be seennear the CAPs_(—)17170 marker. Thus, when sequences for alleles at thislocus were compared between two given lines with differing DM resistancescores, they may have matched at the position exploited for the markerassay, and so both could be defined as the same genotype. However, thesequences of such lines were found in some cases to differ for instanceby up to 3 SNPs at sites near to but not exploited by the particularmarker assay.

Genomic DNA sequences were obtained and utilized to design PCR primersfor detecting single nucleotide polymorphisms, as markers to identifythe presence of the QTLs. The genomic sequences flanking 50 SNP siteslinked to QTLs 1, 2, or 4 on chromosomes 5, 4, and 2, respectively, aregiven in Table 13 (SEQ ID NOs:20-69). These sequences were used todesign sets of 3 synthetic oligonucleotides for each marker, accordingto the GoldenGate® multiplex genotyping protocol (Illumina, Inc., SanDiego, Calif., USA; e.g. see Fan et al., Cold Spring Harbor Symp. Quant.Biol. 68:69-78, 2003; and Gunderson et al., Nature Genetics 37:549-554,2005). These sequences may also be used, as is known in the art, todesign analogous assays, e.g. TAQMAN assays, for marker identification.

TABLE 13 C. sativus genomic DNA sequences flanking the sites of SNP markers linked to QTLs 1, 2, or 4. Chromosome; Marker mapGenomic DNA Sequence (SEQ ID NOs: 20-69). SNP Name position site with polymorphism is given within brackets. NN0223782 2; 22.448TTCATACGCCGTTGCAGCTGAAAGTGGCAACCATTACTTCCAGATCATT ATGACAATAAA[T/C]AACCAAGGCCACCTTCATGCATAAATGGTAAGATAATAGCAAGCTCTATACCTTCTTTTT NN0225385 2; 27.538TCTTGATCAATCCAACTGGGTTGGAACTCAAATTCAAGAAATGGGGTTT AATCACAACCA[T/C]GTTCAATCTCAATTTTCAGATTCCGCCATCCCCCCCACTCCCTATACTCAACCTCCTGAC NN0226670 2; 30.115GGTTTAGATGAAAAGAAGTATGCATTCATGCTTTTGCACAAAGGCATTC CTGGCTTTCAA[A/C]TAGCTGTATCTCTTGCAGGGATATAAATGGATGCAACACTCTTTTCAGTGAAAGAAATCC NN0224124 2; 32.406GGATCATTATTTATCCAACGTATTAGCAAGCTCTAATAGAAACACTTCCTAAAGAACATA[T/C]CGATCCAAATTATATGCTAAGAGAATTACCCAACATTTGAGCAAGTTTACTGACTACAGC NN0246472 2; 38.441TAGAAAACAAGAATGGTTCTCAAAGAACCTACCGACCGAATTGGTTGAGGATGAGATGAC[A/G]AATAGAAACATTAATAATATGGTGGAGGAGGAGGCGATCGATGAACCGTCGCAAAGCATT NN0225358 2; 50.250CCACGAATGGAATCAATGAATTCACGAGCAACCTATAATAAGAGAGTGGAAAACAAAACT[A/G]CTCCTTCACCATCTAAGAAAACAATGTATAAAAATCAGAGAAGGAAAGATAAAAGATACT NN0227700 2; 55.622GAAGAAGAGTGCAACAAGTAAGAGTCTGGCTGGGGGAGTTGAAGTTCATTCATGGATTAG[A/G]TTAACATACGAACTTTGGAGGCGTGCAAAAGACAATCCCCATAACTTGATTACGGGCTTC NN0224617 2; 57.989AAAATATATGATCCTACAACTAAATAAGAGTGCACATGAAGATACCTTATATATAGGAGA[T/C]AATGATGTCGTATGTCCTGCTTGATGATTTCGAAGAACCAGATGACTGAAGAAGGAATAA NN0247695 2; 66.466GGAGTTAATCGAAGGAAGAAAATCAACTCGTCTTAACAGCATCACAATAATCAAATTCTT[T/C]AGGTATACCTTTCTTCTCCTTGTCACTGGACTCATCAGGATCCTCATCGGCGATTTTGCG NN0227242 2; 73.201CTCTGAAGAATTACCGAAGGGGGTCGGAATTTTCTCTATAGCCTGCAATGAGAGGAATAA[T/C]GAGGAATGAAAATGTTGGCTCTGTAGCACATGAATGAAATGCGGATATTCTGCCAAAGGC NN0223824 2; 78.867AGAACAACCCCCAACGTCCCAAAATCACTACATCTCCAACCCTTTCTTCTCCTCATCATT[T/C]AGTTTTAGTCTCTGTTTTGTGGAACTCTCAAATGAAATTGCTTGAATACTCTTGATAAAA NN0223181 2; 82.411ACCAAAAATGAAATAAAATCAGGCTCCACCTTCACCTTGCAGTAATATGATGGCAGTACG[T/C]TGCATTGTAAACCATGTTAATAGAAGAAAAGAA CTGTGAGAAAATACTAGAATTCNN0226638 2; 88.353 CGGATGAGGATTTCAGGTGTTTTTTCTAAACAAATGTTTGCCCTTTAAACATGCGTTTGC[C/G]GATTCTGGTTTATTTGTTTTCGGTTGAT NN0225012 4; 20.649ACTATCCTAATACTACGGAATTGGTGTGGAGAACAGAACAGTTATTTGGTTTGTTCGAAA[A/G]TCTCGACAACTTTCGATCGATCACTCTTGTTAGGGGGTATCCCAACTACATCATAAGCAA NN0228579 4; 25.816ACTCATATTTACAGAAAACTTACTCTAAACCACAAGTCCTTAACAAATATATTTCTGCTT[C/G]CGGCTCTCTTCCTATCATGAAATTTTGCAAGCT ATTCAGAAAATCCNN0226451 4; 27.944 TGCTTCTTGATCTTGCTAATGTAAAGAGATATCACTTACATGAAAGGCTTTCCGAGTCAT[T/C]GTCAATTTCTGCACTGGGAGGCTTTTGGTCATTGCTCTTGAATACCACATTCCCATATTT NN0225088 4; 30.428GAGTTTCCAAAATTGAACATCTTCAATGGACAGAAAGTTTTGAAGTAGCAAGACTTAAGG[T/C]TTCCACTTGGTGTTCCTTATCTAAGTTCTCGAACAATTTGCATTTGATTTCTTAAATATT NN0226219 4; 32.977TAATAGTTACATGATGCTTTATTGCTACTATATATGTTCAGAAATATTATCCAGATGCGA[A/T]ATATTTTCAGACACTGCTGTGAATGTTATTTGGACTAACGAAACTTGTTATTTTGTGCTG NN0247551 4; 35.460GTTTACATGAAAnTATGCACACACCCAAAGATATGTTGATTAAGATGATAAGTTCCCAAG[A/G]ATAGAAGAATTATGTGTGTATTGCTTCTTTGATGTCCAGGAACTAATGAGATTTATCTGG NN0246357 4; 40.294TATAGCCGAGCAACCGAGTCCTTAGATTTGGTTTAAGAGCATCTACGAATAGATGGTCGA[T/C]TGTATGAATGATGAGCAGAAACGCTCTTGAAACGGCTTCnTCACATTTGAACTCCATGCA NN0225551 4; 46.640TGATTTATCTCATGGAGAATACCAATGTGCAAAAGAAACCAAAGGCGTTCTTCTTCATCT[A/G]TGTTTAGCTTCGATATTGTGTGTAGCACTGCAN NNNNNNNNN NN02267324; 49.999 AGAAATTTTTTAAGACAATACTTGATATCCTTTTCACAATTCAGTTCATTCCTCCTATAA[T/C]TGTGATTCCAGCAACGATTAGGAATGATTTCTCAATTCCCACCTTGGTGCACATTTCAAG NN0247689 4; 51.938CTTTATTTTATTTTCAGGTTGCTCTTAAATTTGAGCACAGAAATAGTAAAGG[A/T]TGCAATTATGGTCCTCCATACGAATGGCAAGTTTACAAGTG AGTAACTTTTTGGGTTACANN0247342 4; 54.495 GTACTTGTACCAATATGAAATGTGCACATGCGCTCTTGTCCTAGATAATATGCACAGTTC[A/T]CCTTAAAAGCTAAGCATAAGCCACAAATCCAAGAACCAATGTAACCAAAACAGTATGGGA NN0224702 4; 62.385CTGTTGCCTATGCAAAGCATTTATACAGCTTCATTCTGCCATTTTAACGAATGTTCACTG[A/T]AGTACCTGAGATGGCTTCCAAAACTGTCTTGTAAGGTGTGCCTCTCATCTGCATCTGTCT NN0225482 4; 66.478TCTTTTTATACTTTTCTGATCTTGTAAAGTTTAAGGCTTTCAACTGGTGTAGTGTAGTCA[A/C]CAGAAGTCTTTTTATAATTGTTACACTTTAATT TGATAGGAAAGTGTTTCTTTAANN0224538 4; 67.148 GCTAGTATATCTATATATCTTTTTGAGAGCTTTATGATAATTATCAAATGAAGATTCTAG[C/G]TGTGTGATCAAGAGAAGATTCCTAGCCTACCTCTTAGCTCTTTTAAAAATTGTGGTAGTT NN0247543 4; 71.973TAAnTTTGTTTTCACATTTnTTTnCAAATAATATATATCTTACAGTTTAGGAGGCTTCTT[T/G]CACCATTACATATAAAACATATGGAACAAACATTACCTCTAAATAACCAATAGACTTTTA NN0224041 4; 75.834TCTTTAAAAGAAGCTTGAAAGATGAAGAAATTGCAAAATTTCAATCATTTCGATCCCTCC[A/T]CTCACCTGAAAAAGTGGTTAATTCTGATGATTTTAGATCATGGTCCATTGATCCCTCAAT NN0228853 4; 78.923GTCCATACTACCATAATTGATAATACTAAGTACCCTGAACCTTTGAGATTTGAATCTTTC[T/G]ACCCCACAGGTTGTTAAACACAAATACGAGTAACAAAGATAAGATTTGAACTCAGCCTTT NN0227762 4; 85.871TTGGTTCCTGATCCAGAAGAAAAATGGTTGATTTCCTTATGATTCTTTTCAGTAAGGTGG[A/G]ACAATCTTGCTCCAGGTCCTTCGTCATAAAACACCCCCCTCCAAGGAAGAGAAACCCCAA NN0227587 4; 87.395ATTTTTAGATGTGAGCCTATTTATGTGCTCTTAACTTCTCTTTTAGTAATGGTGGAAATG[T/C]AATTTGTTTATGAGCAATGGTATCGTGAATAAATGGTTGGATACATAATAGCTTTGCTTG NN0228457 5; 25.235GTTGGGCTTTTTTGTTGTATTATTTCTTTTTGTCGTATTATGTATCTAAAGGCAGATGAA[A/G]ACCTGGATCATATTCTTTGGCAATGTGATTTTGCGTTGGTGTTTGGGATTCCTTTTTCCA NN0246356 5; 30.032TTAAACAATTGACCCAAAAnTTAAAGTTGATGGGTAAAGGTAAAACTAACATTATATCAC[A/G]CAACATTCCCTCATTTGTAGACTTGAAATATGTAGAAAAGCCCATTAGTGAAAATGAATA NN0246332 5; 33.371TATTTTGGTGCTAACCGAATAGTTTGAGAGGGAGAAATAGGAAACGATGACAACnCCATG[A/G]ATTGGGGGAAGTGTTCGACATTTGAAAAGGAGAATAAATCAATTAAAATTATGCATTCAA NN0223399 5; 38.201ACACAGTATTGATAATTTCCAAATCAACACTTGGAATATTTTTGTTAAATATGCACGACA[A/T]ATTTGGTTCGTTATTGGTCCATGATGGTAATCTATACTTCATATGAAATTCTTATCTCTA NN0223689 5; 41.014TATATGTTGCTAATGTGCTATTTTTAAAGAAAAAATAAAGGCCCCATTTGGTCATGGTTT[T/C]CTCTCAATTGTTCAATTGTACTTTCCACATTATTTGAATAAGCAATCCAACTTTTACCTA NN0247731 5; 44.780TGAAGAAGAGCCTTTATTGCGTCATCTGGAACCTCCTTTATCCATTTATCTTGAATTGGT[T/C]GGTCATGAACACACCTTTACCATTTATTATTCAATGCCATGATTGTCTTACACAGGTGTG NN0226166 5; 49.540AGTCGAGTTTTGAGCCACTCATAACCCAGTTGAATGCTTTTAACCTTGTAGTCACTAACA[C/G]AAACACAGTGGAATGGTAGTTGAAGTAGGGTCATTTTGGCTAATTCATTGCATGTTGTTA NN0225480 5; 52.743TGTCCAAAAGAAATATATGCTCAAACTGTGCCTCAATCACCGTCTTCTCTCCAATTTTCC[T/C]ATATCATTCTCGATTATTACTTTCCTCTATTTCATTTAGTTTCTAATAATTAATAACGGC NN0246411 5; 55.649CCGAGGCGACCTCCGGCTTGCCCGTTACAAGTTGGGAGTTGGGCCTTGGGCTCTCGACGT[A/G]GGCCGAGAGTCCATTGCTCCTAAAGATTGGTGGGCACCGCGTnAGGCTGGTTCACAGAGC NN0227759 5; 59.710CTTCTAGCCAAAATAATTCCATTTTTGTTTCACAAAATGAACCTGTTGGAGGGAAGACCA[C/G]TGCGCTACCCATCCCAAATTCAGATTCTGGAAATATGGCCAAAGATCATTCATTGAGGTC NN0247348 5; 61.450ATGGAGCTTAGAGAAAACGACCTAGAAAnTATTATCCTTATTCATGTCGAACGGGCTGTC[T/C]GGTAAGATTAGTTGAGGTGCGCATAATACCTGGACACTCACAAATATGnnnnAAAAAAAn NN0228465 5; 68.021ATATGGAATATTTGACAACTAATTAGTTACTTCTAAAACTGCAATAGCAGTCACGTTAGT[C/G]TCCCCCAGGGAGAGAAGAAAATACTAGAATTTGTGAGCCACTGTTAATAGATTCAAAATA NN0247786 5; 71.424TTAGGTTCCCGCTATTGCAATACCAACAAGGCATGAAGGCTTT[C/G]GCCACAATGTGAAATCATCAACAGTTACTATGAACAATGAATTCGCTAAC AGGAAAnCGT NN02266455; 74.784 TCTAATTCGATGAGTTTGTCTGAATTCCCAAGTAAGAACCAAAGTTCCATTCATTCTCTC[A/G]AGCACAAACCTTTCCACCAAAACATAACACCTAAATCTCTTCCACATTCCCTTTCCTTTA NN0223809 5; 76.617GGAGATTGTTGCACGCCTGAAGAAAGCCTTCAGAATTACCATTAAGACTTCCCAACTCTG[T/C]CTGCATATTGTCAAGAAAGCTAGAGATTTTCTCAGAAGCTACTTTAATGGCAGGGCCCTC NN0227071 5; 78.303AGGAAAATACCTTCCAATAGAAAACATGGTAAATGCAATAAGCATCTAGTTCATCCAATT[C/G]CAAGGGTAATGGCTAGTTCAAGATGGAACTAAAACTCTCAGAGTGGTGTGTGCAATCCTG NN0226870 5; 80.340GGTTACAGAGTCCAGAGCGATCAATGGAGATTGTGGATGTTGAAGGTTCAGAAGAGAAGG[A/G]TCGTTGATTAAGGAGGTCTTCATTCTCCAGGTGCTGAACGACAGTACCTTCCGGAATCTG NN0228148 5; 82.273ATGATCCTCAATTCATACCATATTATTGTATGAGCTAAATGTGTATAAAGAAAAGGAAAG[T/C]TATAGAAGGATGGATTCGTACATTAACACAAAGGATAAAAACTGCAAACTTATTTATATA

Plants containing a DM resistant donor allele(s), including linesGSP33-1094GY and GPN33-1093, among others (see Tables 1-8, and 14) wereselected for further breeding. Depending on the breeding strategy,plants selected for further breeding can be either homozygous orheterozygous for a donor (resistant) allele.

TABLE 14 Line Descriptions- Selected Agronomic and Disease ResistanceTraits for Additional Lines with Introgressed DM Resistance andComparison Lines. Lines with a high level of DM-resistance SeedFlowering Scab PM DM Line code Source Type pattern ParthenocarpicPlantbitterfree CMV (Cladosp. Cuc.) (Sphaerotheca f.) (Pseudop. Cu.)05-346 NJ05 Pickling, GY Yes Yes 1 1 2 2 854-3 parth. spined GSP33- NJ05Pickling, GY Yes No 2.5 5 1 2 1094GY 696-4 parth. smooth 01-349 VJ02Pickling, GY Yes Yes 1.5 1 1 2 322-6 parth. spined GPN33- VJ02 Pickling,GY No No 3 5 2 2 1093GY 55-3 poll. spined Lines with a high level Markergenotypes of DM-resistance DM Smooth/ Spine Mean Spined color CAPs_ENK59CAPs_ENK60 CAPs_17179 CAPs_17170 CAPs_18229 CAPs_17563/66 score SpinedWhite B B B B A B 1.8 Smooth xxxxx B B A A A B 2.0 Spined White B B B BA B 2.3 Spined White B B A A A B 1.8 Lines susceptible to DM Line SeedFlowering Scab PM DM code Source Type pattern ParthenocarpicPlantbitterfree CMV (Cladosp. Cuc.) (Sphaerotheca f.) (Pseudop. Cu.)05-110 VJ05 Pickling, GY Yes Yes 2 1 1 4 110-3 parth. smooth 01-714 NJ05Pickling, GY Yes Yes 2 1 2 4 684-4 parth. smooth 05-779 VJ05 RiesenschalGY Yes Yes 5 5 5 5 273-4 parth. Lines susceptible to DM Marker genotypesSmooth/ Spine DM Spined color CAPs_ENK59 CAPs_ENK60 CAPs_17179CAPs_17170 CAPs_18229 CAPs_17563/66 Mean Smooth xxxxx B B B B A B 3.6Smooth xxxxx B B B B A B 4.1 Smooth xxxxx A A A A A A 5.0

Line 05-346 was found to display strong vigour, with dark green fruitcolor and cylindrical fruits with a length/thickness ratio of 3.2. LineGSP33-1094GY was found to display strong vigour, with fruits rather long(length/thickness ratio of 3.3/3.4), and leaves somewhat upright withlittle leaf tendency. The skin of the fruit was somewhat rough. Line01-349 was found to be productive, with good fruit shape. Fruit fleshwas firm, and fruits displayed an average length/thickness ratio of 3.3.Line GPN33-1093GY displayed strong vigour. Leaves were somewhat curled.Its fruits were slightly spined, but the density of spines was very low.The average length/thickness ratio of fruits was 3.3.

Example 5 Creation of Additional Populations for Mapping DM ResistanceLoci

To identify loci underlying cucumber resistance to the Downy Mildewpathogen (Pseudoperonospora cubensis), two additional mappingpopulations were generated from crossing each of two susceptible parents(API-45-5312-MO and VJ03-68-06) by the same resistant line (PI197088).From each of these crosses a single F₁ plant was self pollinated tocreate two F₂ populations. These were coded as “API” and “VJ”populations, from the crosses API-45-5312-MO×PI197088 andVJ03-68-06×PI197088, respectively. Beginning at the F₂ generation, asingle-seed descent procedure was used to create ˜200 F₅ families ineach of the two populations. The process involved self pollination of˜250 F₂ plants in each population to create F₃ families. From each F₃family, a single plant was self pollinated to create F₄ families. Fromeach F₄ family, a single plant was self pollinated to create F₅families. When the F₄ plants were growing, tissue samples were collectedfrom each plant. These samples were used for DNA isolation and markergenotyping. Multiple plants from each of the F₅ families derived fromeach F₄ plant were tested for reactions to Pseudoperonospora cubensis inreplicated, multi-location pathology (i.e. diseaseresistance/susceptibility) trials (see Example 6).

Example 6 Analysis of Cucumber Plants in Additional Populations for DMResistance

Marker genotypes for each F₄ plant from the mapping populationsdescribed in Example 5 were then analyzed against the performance of theF₅ progenies in DM disease resistance/susceptibility trials, in a parentgenotype vs. progeny performance manner. Cucumber plants from thesepopulations were grown in multiple locations including Turkey, Thailand,France, U.S.A., Netherlands, and India, and subjected to bioassays undergreenhouse and/or field conditions, to determine the level of resistanceto Downy Mildew disease caused by P. cubensis. Resistance testsperformed in some locations utilized the protocols described above, inExamples 1-2. Resistance tests performed in Nimes, France and in Turkeyutilized a similar protocol, however, for instance, a P. cubensisisolate found naturally at Bortepe, Antalya, Turkey was used, whiletests in the Netherlands and in Thailand utilized P. cubensis isolatesalso obtained locally. Tests were performed on the mapping populationswith a panel of cultivars selected from the group including: SMR58,Maram and Corona (highly susceptible); Poinsett 76, Adefem (partiallyresistant); Sweetslice (intermediate resistant); and PI 197088 (highestlevel of resistance) as controls. Inoculations were performed in thegreenhouse using freshly sporulating detached leaves, about 6 days to 1month after planting. Up to three evaluations of cotyledons and 1^(st)and 2^(nd) leaves were performed about 6-30 days after inoculation. Anexemplary disease rating scale is defined as follows:

1: True leaves or cotyledons show no symptoms.

2: Leaves or cotyledons are green, possibly a few chlorotic spots.

3: Necrotic spots are not confluent; less than 30% of cotyledon area isaffected; leaves have larger chlorotic spots with necrosis at center.

4: 40% of cotyledon area is necrotic; less than 25% of leaf area isnecrotic.

5: 50% of cotyledon area is necrotic; less than 50% of leaf area isnecrotic, not coalescent.

6: 60% of cotyledon area is necrotic; coalescent necrotic area on 25% ofleaf surface.

7: 70% of cotyledon area is necrotic; coalescent necrotic areas cover50% of leaf surface.

8: On cotyledons and leaves, only tissue near petiole is still green.

9: cotyledons and leaves are completely necrotic.

Representative levels of disease on control lines and in a mappingpopulation are shown in FIGS. 3-4.

Example 7 Methods for QTL Mapping Analysis of API and VJ PlantPopulations

QTL mapping analysis was performed on the data obtained from diseasetests on the two additional mapping populations derived from crosseswith PI190788. For the API population, 971 mapped SNP markers across 174genotyped and phenotyped lines were utilized. For the VJ population, 964mapped SNP markers across 168 genotyped and phenotyped lines were used.Exemplary genetic markers are listed in Table 13, and FIG. 5schematically illustrates the positions of these markers which localizeto chromosomes 5, 4, and 2. QTL-mapping analyses were performed in QTLCartographer v1.17 (Basten et al., 1994; and Basten et al., 2004). Theprograms used were: LRmapqtl, SRmapqtl, MImapqtl, MultiRegress,JZmapqtl. All two populations×6 locations (=12 datasets) were analyzedindependently.

Interval mapping was performed at 1 cM intervals using LRmapqtl. Theparameters used for the program were: M=3; d=1; c=0; r=0. StepwiseRegression Mapping with forward-selection followed bybackward-elimination was performed using SRmapqtl. Markers were includedin the model at P<0.001 at the forward-selection step and excluded fromthe model at P>0.001 for the backward-elimination step. Markers within10 cM of markers already in the model were excluded from furtheranalysis. The parameters used for the program were: M=2; F=0.001;B=0.001; u=10.

Multiple Interval Mapping was used to search for multiple QTL inmultiple intervals. JZmapqtl was first used to estimate genotypefrequencies at 1 cM intervals. The parameters used for the program were:I=30; M=9; d=1. MultiRegress was then used to estimate the initial setof putative QTLs using stepwise regression testing for all 1 cMintervals for both additive and dominance effects and a QTL is includedin the initial model at P<0.0001. Test sites within 20 cM of a putativeQTL already included into the model are excluded from further analysis.The parameters used for the program were: c=0; S=0.0001; w=10; u=20;I=30. MImapqtl was used to test and eliminate non-significant putativeQTLs from the initial model identified by MultiRegress (−I=sMPrTseC). Atthis step, the maximum number of QTL is limited to 5 (−q=5), and aputative QTL is deleted if the change in information criterion is<13.825 (corresponding to 3 LOD). MImapqtl was then used to refine theQTL position (−I=sMPRtseC), and search for any new QTL (−I=sMPrtSeC). Atthis step, the maximum number of QTLs was limited to 5 (q=5), and aputative QTL would be added if the change in information criterionis >13.825 (corresponding to 3 LOD). A search for epistatic interactionsbetween putative QTLs (I=sMPrtsEC) was also carried out. At this step,an epistatic term would be included in the model if the change ininformation criterion were >0.000. All other analyses including analysesof QTL Cartographer results were performed using R (R Development CoreTeam, R: A Language and Environment for Statistical Computing. RFoundation for Statistical Computing, Vienna, 2009). For the assessmentof the modes, disease ˜QTL1+QTL2+QTL4.

Example 8 QTL Mapping Results for API and VJ Populations

A total of 23 apparent QTL intervals (2-LOD interval 5 LOD QTL peaks)were identified across the 2 population×6 location experiments. Many ofthese overlap between populations and/or locations, and three QTLs,termed QTL 1, QTL 2, and QTL 4, mapping to chromosomes 5, 4, and 2respectively, were identified for further analysis. Markers utilized,along with their positions, are given in Table 13, and approximate mappositions for the QTLs are also given in Table 15.

Of particular interest are QTL 1 on chromosome 5 and QTL 2 on chromosome4, which were identified in both mapping populations grown in at leastthree of the six geographic locations, suggesting the robustness ofthese two QTLs in different genetic backgrounds and environmentalconditions. The average individual additive allelic effects are 0.62(0.32-1.26) at QTL 1 and 0.62 (0.47-0.93) at QTL2. Therefore, anindividual plant with both resistant alleles at both QTLs is likely tohave a reduction in disease rating of 0.62×2+0.62×2=2.48. Individually,the average amount of phenotypic variation explained by each of thesetwo QTLs is 24% (QTL 1) and 21% (QTL 2), with the remaining 76%-79%attributable to other genetic effects and environmental (non-genetic)effects.

A negative additive effect (i.e. a resistant genotype with higherdisease score) was identified at chromosome 2, position 21.8-73.8cM inthe VJ population grown in Turkey, corresponding to QTL 4. The same QTLwas also identified in this same VJ population grown in Nimes andThailand, but the additive effect was positive. Thus, the negativeadditive effect identified in the Turkey dataset is likely not anartifact but rather suggests the presence of genotype×environmentaleffects.

TABLE 15 Summary of interval mapping results for two mapping populationsacross 6 locations. Approx. Interval Mapping Number of Additive QTL #Chr (cM) Population Locations Effects R² 1 5 22.6-81.0 (58.4) Both 60.32-1.27 12-39% 2 4 37.7-87.6 (49.9) Both 3 0.47-0.93 13-28% 4 221.8-73.8 (52.0) VJ 3 −0.49-0.61  12-20%QTL intervals shown in Table 15 were identified by Interval Mapping.Each interval is labeled by a unique identifier (QTL #) with chromosomallocation on a consensus cucumber genetic map (e.g. Ren et al., 2009)indicated. QTL intervals may be identified in one or both of the API andVJ mapping populations and in one to six of the six geographic locationswhere testing was done. The range of additive allelic effects andproportion of phenotypic variation attributable to the corresponding QTL(R²) are also indicated.

Exemplary SNP markers corresponding to three QTLs (termed QTLs 1, 2, and4) which were identified are listed in Table 13. These QTLs map,respectively, to chromosomes 5, 4, and 2. Of 23 putative QTL intervalsidentified by the interval mapping approach, examples of which are shownin Table 16 and 21, were also identified by a stepwise regressionapproach using forward-selection-backward elimination. The two putativeQTLs which did not corroborate did not correspond to either QTL 2 onchromosome 4 or QTL 1 on chromosome 5.

To estimate the joint effects of multiple QTLs at multiple intervals,multiple interval mapping was performed for all datasets. The resultsagain identified QTL 1 (mapping to chromosome 5) and QTL 2 (mapping tochromosome 4). QTL 1 was identified in both the API and VJ locationsgrown in most (5/6) of the locations (Table 17), suggesting this QTLinterval is robust in view of both genetic backgrounds and environmentaldifferences. Conversely, QTL 2 was identified in the API populationgrown in three of the locations and in the VJ mapping population grownin Turkey (Table 17), suggesting that the effect of this QTL may bedependent on the genetic background.

In contrast to QTL 2, QTL 4 on chromosome 2 was identified from the VJpopulation grown in three of the six locations and in the API populationgrown only in Turkey. This suggests that while QTL 2 may be specific tothe API background, QTL 4 may be specific to the VJ background.Interestingly the allelic effect of QTL 4 becomes negative when QTL 2 isalso in the model (in both mapping populations grown in Turkey),possibly suggesting (1) an opposing effect between QTL 2 and QTL 4, or(2) that the negative effect at QTL 4 is specific to environmentalconditions found at the testing site in Turkey. Assessment of the model:disease ˜QTL1+QTL2+QTL4 for the other datasets suggests all three QTLsshould have positive additive effects (also see Table 18), implying thesecond explanation is more likely.

Epistatic interactions were only identified in the two Turkey datasetsand only additive-by-additive effects were identified. The interactionswere between the major QTL intervals QTL1, QTL2, and QTL4. Becauseepistatic interaction is only identified in the Turkey data and becauseof the negative effect of QTL4 in these dataset, at this point, it isunclear whether the negative additive×additive effects between QTL1 andQTL2 is also Turkey-specific.

TABLE 16 QTLs identified by Multiple Interval Mapping for the twomapping populations grown in six locations. QTL1 × QTL2 × PopulationDataset Location QTL1 QTL2 QTL4 QTL2 QTL4 R² VJ 1 Turkey 0.61 0.47 −0.46−0.23 0.67 2 Nimes 0.58 0.36 0.52 3 Thailand 0.59 0.65 0.29 4Netherlands 0.16 5 India 0.47 0.36 6 Felda 1.28 0.33 API 7 Turkey 0.470.57 −0.45 −0.22 0.23 0.56 8 Nimes 0.69 0.49 0.66 9 Thailand 0.74 0.900.47 10 Netherlands 0.32 0.15 11 India 0.52 0.32 12 Felda —Among a total of eight unique QTL intervals identified by at least oneof the experiments, QTLs 1, 2, and 4 are shown in Tables 16-17, andtheir corresponding genetic locations are indicated in Table 15. InTable 16, the last column, R², is the estimated proportion of observedphenotypic variation explained by the genetic model. For datasets 2, 4,5, and 8 of Table 16, minor putative QTLs were included in the analysisof the proportion of observed variation explained by the QTLs (column)in the model of the experiment (row) and the numeric values are theestimated additive allelic effects (additive×additive in the case ofQTL1×QTL2, or QTL2×QTL4).

Genomic sequences from cucumber DNA clones containing RAPD Geneticmarker CAPS_ENK59, among others, such as was used to identify theDM-resistance QTL described in Example 3, Tables 10-11, and FIG. 1, werealigned to a cucumber genomic map to determine their locations withinthe cucumber genome. Sequences were aligned using Sequencher (Gene CodesCorp., Ann Arbor, Mich., USA), and found to fall within 13 contigs (11loci completely assembled, and 1 locus assembled onto two contigs).These sequences were then aligned onto a cucumber genomic physicalsequence scaffold map using BLAST and GMAP (Genentech, South SanFrancisco, Calif., USA), with the RAPD marker positions (converted toCAPS) being placed on the integrated physical-genetic map based onsequence similarity. Additionally, the CAPS_ENK59, UBC12-1200, andAL10-850 markers, which were found to be linked with the originallydefined QTL in the previous mapping experiments based on QTL analysis oflines derived from a Lucinde×PI197088 cross as described in Example 3,were also followed in the new API and VJ mapping populations todetermine where they localized relative to the newly identified QTLs.Both approaches resulted in the finding that the originally identifiedQTL of Example 3 co-localizes with QTL 1 and maps to chromosome 5, asshown in FIG. 5. Marker NN0246677 maps around position 43.8 or 47.8respectively, of the respective API- and VJ-derived chromosome maps ofFIG. 5, well within the genetically defined QTL. Although this is somegenetic distance from CAPS_ENK59 which was utilized in the initial work,the markers identified as co-segregating with this QTL (i.e. QTL 1) wereidentified in different mapping populations, and in particular, theinitial work genotyped fewer markers and had fewer replications ofphenotyping trials than were utilized subsequently with the SNP markerssuch as NN0246677. Thus, the original mapping population included fewerrecombination events, allowing for more distant markers to be morefrequently retained in a linkage block that showed significantassociation with a DM-resistance QTL. The QTL analyses in differentpopulations had different levels of statistical significance as well,resulting in somewhat different map positions.

Additional putative QTLs specific to various combinations of mappingpopulation and geographic location were also identified, but the lack ofreplication across location and/or mapping population potentiallysuggest these QTL contribute too little to the overall phenotypicvariation for replicable detection.

A summary of the interval mapping results is shown in Table 17, with 2LOD intervals at 4-LOD QTL peaks for each of the 2 population×6 locationexperiments. Chr: chromosome; From: left position of the 2-LOD intervalin cM; To: right position of the 2-LOD interval in cM; LR: likelihoodratio between the alternate (H3) and null (H0) hypotheses, whereH3=presence of a QTL with additive and dominant allelic effects at thetest site and H0=no QTL at test site); a: additive allelic effect; d:dominant allelic effect; R²: proportion of variation explained by theputative QTL at the test site, SR: whether the same QTL was detected bythe Stepwise Regression approach; MIM: whether the putative QTL wasdetected by the Multiple Interval Mapping approach.

TABLE 17 Interval mapping results. Population Location Chr From To LR ad R² SR MIM VJ Nimes 2 21.77 66.48 21.53 0.41 0.23 0.12 Y Y VJ Turkey 239.45 48.76 36.46 −0.48 0.14 0.20 Y Y VJ Thailand 2 46.76 73.80 28.790.61 −0.58 0.16 Y Y VJ Turkey 4 37.65 73.98 32.79 0.47 −0.02 0.18 Y YAPI Nimes 4 46.65 71.65 38.00 0.56 0.18 0.22 Y Y API Turkey 4 55.8279.93 43.23 0.53 −0.27 0.22 Y Y VJ Thailand 4 57.50 87.57 21.85 0.58−0.08 0.13 Y — API Thailand 4 74.98 81.98 51.79 0.93 0.25 0.28 Y Y APITurkey 5 22.58 56.40 27.88 0.43 −0.42 0.16 Y Y API India 5 26.64 30.3159.67 0.52 0.15 0.32 Y Y API Netherlands 5 33.84 77.66 24.21 0.32 −0.130.15 Y Y VJ Thailand 5 40.37 55.81 20.24 0.55 0.22 0.12 Y Y API Thailand5 44.71 56.40 34.46 0.79 0.55 0.22 Y Y API Nimes 5 44.71 55.40 70.720.71 0.04 0.39 Y Y VJ Nimes 5 46.19 55.81 45.75 0.61 0.13 0.25 Y Y VJTurkey 5 53.10 80.70 51.56 0.57 0.29 0.27 Y Y VJ India 5 53.10 80.9535.43 0.45 0.13 0.19 Y Y VJ Felda 5 76.71 80.95 58.81 1.26 0.48 0.33 Y Y

Linear Regression was also utilized to calculate coefficients of diseaseexplained by the model QTL1+QTL2+QTL4 (see Table 18). Unlike QTLCartographer, which always compares the change in phenotype against theresistant line, the coefficient values of Table 19 correspond directlyto the change in disease rating from 1 (resistant) to 9 (susceptible).Thus a positive coefficient implies an increase in susceptibility.

TABLE 18 Coefficients of disease obtained using linear regression.Population Location Intercept QTL1 QTL2 QTL4 VJ Turkey 8.6 −0.56 −0.410.45 VJ Thailand 8.8 −0.28 −0.51 −0.46 VJ Nimes 8.9 −0.51 −0.29 −0.39 VJNetherlands 6.3 −0.12 −0.03 −0.05 VJ India 9.6 −0.44 −0.15 −0.13 VJFelda 9.4 −1.26 −0.20 −0.08 API Turkey 8.1 −0.40 −0.54 0.40 API Thailand7.0 −0.55 −0.65 0.06 API Nimes 6.9 −0.54 −0.37 −0.01 API Netherlands 5.5−0.22 0.05 −0.02 API India 9.2 −0.38 −0.18 0.09 API Felda 6.6 −0.05−0.54 0.31

Example 9 Alignment of SNP Markers to SSR-Based Cucumber Genetic Map

For mapping of QTLs relative to additional markers, the SNP markers ofTable 13 were aligned with SSR markers which were used by Ren et al.(PLoS ONE 4:e5795, 2009; doi:10.1371/journal.pone.0005795) by aninformatics approach. First, the 16-25 bp primer sequences defining theSSR markers of Ren et al. were aligned with corresponding cucumberscaffold sequences (genomic fragments) using BLAST. Perfect alignmentscovering entire primer sequence pairs (i.e. both forward and reverseprimers) were filtered to remove any apparent perfect alignments whichdid not suggest the forward and reverse primers properly facing eachother, and the remaining scaffold positions were merged with theSSR-based map information. 574 SSR markers were putatively mapped to 591scaffold genomic fragment sequences, since, as expected, some SSRsequences mapped to >1 scaffold location. Using a size differencethreshold of less than or equal to 50 bp for the difference between thereported forward and reverse SSR primer locations and the mappeddistance as indicated by the scaffold sequences, 550 SSR markers (with566 mapped locations) were considered to be reliably mapped. This SSRdata was then sorted with Scaffold IDs, and was merged with knownpositions of other available internal markers. Finally, inconsistentmarker positions were noted, and remaining SSR genetic positions wereanchored to a consensus genetic/physical map that had been built usingSNP markers and genomic scaffold sequences. Table 19 lists map positionsof SNP and SSR markers of chromosomes 2, 4, and 5, which may be directlycompared with the genetic map of Ren et al. (2009). SSR and other publicmarkers listed in bold in Table 19 map to the identified QTL regions, orare about 10 cM or less from such a region, and are expected toco-segregate with DM resistance. The mapping of SNP and SSR markers aslisted in Table 19 allows for identification, using a publicly availablegenetic map, of additional (e.g. publicly available) genetic markersthat map to a genomic region linked to Downy mildew resistance. In Table19, the DM resistance QTL of chromosome 2 is defined as spanning theregion defined by SNP marker NN0223892 (map position 22.3), to SNPmarker NN0226638 (map position 88.4). The DM resistance QTL ofchromosome 4 is defined as spanning the region defined by SNP markerNN0227442 (map position 20.6), to SNP marker NN0227285 (map position87.4). The DM resistance QTL of chromosome 5 is defined as spanning theregion defined by SNP marker NN0226553 (map position 25.3), to SNPmarker NN0226391 (map position 82.1).

TABLE 19 Genetic map positions of SNP (“NN0 . . . ”) and SSR markers ofcucumber chromosomes 2, 4, and 5, from alignment of SSR markers ontoconsensus scaffold-based genetic map. See also FIG. 5. Marker NameChromosome # Map position NN0226713 2 0.0 NN0246869 2 1.3 NN0227534 21.5 NN0228688 2 1.5 NN0224222 2 1.6 NN0225653 2 1.6 NN0227319 2 1.6NN0247117 2 1.6 NN0223201 2 1.6 NN0246545 2 2.8 NN0247432 2 2.8NN0225708 2 3.3 NN0224023 2 3.3 NN0225478 2 3.7 NN0225481 2 3.7NN0247128 2 3.9 NN0247110 2 4.2 NN0228683 2 4.6 NN0247397 2 4.6NN0229107 2 5.0 SSR11952 2 5.9 NN0227776 2 5.9 NN0225556 2 5.9 NN02251492 5.9 NN0247714 2 6.9 NN0226409 2 7.0 NN0247374 2 7.0 NN0225817 2 7.2SSR21090 2 7.5 NN0228345 2 7.5 NN0224358 2 8.1 NN0224778 2 8.1 NN02475912 8.8 NN0246816 2 9.7 NN0226190 2 10.2 NN0225911 2 10.2 NN0224313 2 10.5NN0224822 2 10.7 NN0225831 2 12.1 NN0224132 2 12.1 NN0224585 2 12.2NN0224551 2 12.2 NN0226478 2 12.2 NN0226856 2 12.2 NN0228321 2 12.2NN0225656 2 12.2 NN0246405 2 12.3 NN0247003 2 12.8 NN0226751 2 12.8SSR00684 2 13.0 NN0229051 2 13.0 NN0247367 2 13.0 SSR16226 2 14.2NN0225537 2 14.2 SSR03070 2 14.2 NN0225564 2 14.2 NN0246551 2 14.5NN0224243 2 14.5 SSR00204 2 14.5 NN0223995 2 14.5 NN0246378 2 14.6NN0228099 2 14.9 NN0227372 2 15.5 NN0246337 2 19.0 SSR00289 2 19.7NN0227840 2 19.7 NN0227646 2 20.0 NN0228476 2 20.3 NN0224590 2 20.8NN0228237 2 20.8 NN0247575 2 20.8 NN0227530 2 20.8 NN0227574 2 20.8NN0224794 2 20.9 NN0224612 2 21.2 NN0224686 2 21.2 NN0224467 2 21.3NN0228820 2 21.3 NN0225896 2 21.8 NN0223892 2 22.4 NN0223782 2 22.4NN0227357 2 22.7 SSR22083 2 22.7 NN0227327 2 22.7 NN0247569 2 23.9NN0223579 2 23.9 NN0226747 2 24.3 SSR23832 2 24.9 NN0226337 2 24.9NN0227615 2 25.4 NN0226084 2 25.4 SSR23220 2 25.4 NN0226279 2 26.5NN0224105 2 26.5 SSR23420 2 26.5 NN0225385 2 27.5 NN0246831 2 28.8NN0228374 2 28.8 NN0223545 2 29.2 NN0224897 2 29.2 NN0226707 2 29.5NN0226670 2 30.1 SSR01374 2 30.1 SSR22338 2 30.1 NN0223657 2 30.1NN0247784 2 30.7 NN0225380 2 30.7 NN0223661 2 30.7 NN0247123 2 30.7NN0228011 2 32.1 NN0228058 2 32.1 NN0224124 2 32.4 NN0246849 2 33.5NN0226619 2 33.7 NN0247070 2 34.7 NN0226477 2 35.3 NN0223707 2 35.5NN0227659 2 35.5 NN0227441 2 35.5 NN0223443 2 35.5 NN0223203 2 35.5NN0226267 2 35.7 NN0224422 2 36.1 NN0227137 2 36.6 NN0246472 2 38.4NN0224359 2 38.4 NN0228139 2 38.7 NN0227805 2 43.5 NN0247413 2 43.5NN0223478 2 43.8 SSR02569 2 43.8 SSR07108 2 43.8 NN0227538 2 43.8NN0247197 2 44.4 SSR11512 2 44.4 NN0226250 2 45.0 NN0224063 2 45.3NN0228486 2 45.3 NN0224233 2 45.3 NN0223285 2 45.3 NN0223271 2 45.3SSR00218 2 50.3 NN0225358 2 50.3 NN0224573 2 51.6 NN0226986 2 51.6NN0224027 2 51.7 NN0228028 2 51.7 SSR17814 2 52.8 SSR12083 2 52.8NN0225190 2 52.8 NN0246702 2 53.3 NN0224844 2 53.9 SSR16941 2 55.5SSR11468 2 55.5 SSR22227 2 55.5 NN0227695 2 55.5 NN0227700 2 55.6NN0228748 2 56.1 NN0246318 2 56.4 NN0247210 2 56.4 SSR02634 2 56.4NN0223380 2 56.4 NN0223398 2 56.4 NN0247233 2 56.9 NN0246696 2 57.4NN0223463 2 57.6 SSR23732 2 57.6 NN0223494 2 57.6 NN0223465 2 57.7NN0224617 2 58.0 NN0224658 2 58.3 SSR05492 2 59.5 NN0246763 2 59.5NN0224594 2 60.9 SSR02322 2 66.5 SSR07278 2 66.5 SSR11909 2 66.5SSR16135 2 66.5 SSR22653 2 66.5 NN0247695 2 66.5 SSR00009 2 67.0SSR06913 2 67.0 NN0227962 2 67.0 NN0226717 2 67.1 NN0247305 2 67.2NN0226038 2 68.0 NN0225776 2 68.5 NN0247544 2 69.7 NN0227412 2 69.8NN0247350 2 69.9 NN0247798 2 70.0 SSR13131 2 70.0 NN0228560 2 70.0NN0226406 2 72.4 SSR21734 2 73.2 NN0227242 2 73.2 SSR00507 2 73.7NN0247636 2 73.7 NN0226006 2 73.8 SSR15873 2 74.6 NN0225709 2 74.6NN0224913 2 74.6 SSR12227 2 76.3 SSR18362 2 76.3 NN0228448 2 76.3SSR21655 2 78.0 SSR05420 2 78.0 SSR19851 2 78.0 NN0225027 2 78.0NN0225018 2 78.0 NN0227692 2 78.5 NN0247139 2 78.5 NN0223317 2 78.8NN0223824 2 78.9 NN0223452 2 79.5 NN0246284 2 79.7 NN0246771 2 79.7NN0223209 2 79.7 NN0223221 2 79.7 NN0226547 2 79.8 NN0247618 2 80.3SSR01253 2 80.5 NN0228741 2 80.5 NN0226471 2 80.5 NN0226327 2 81.0NN0226885 2 81.6 NN0246708 2 81.6 NN0228685 2 81.6 NN0228974 2 81.7NN0246681 2 82.1 NN0226957 2 82.4 NN0223181 2 82.4 SSR21276 2 83.0NN0227214 2 83.0 NN0225257 2 84.4 NN0223574 2 85.8 NN0226638 2 88.4NN0226673 2 88.5 NN0228750 2 88.6 NN0246924 2 90.4 SSR30665 2 91.5NN0224981 2 91.5 NN0224804 2 91.5 SSR13754 2 96.1 SSR16462 2 96.1NN0223703 2 96.1 SSR16028 2 96.1 NN0247710 4 0.0 NN0247539 4 0.0NN0247697 4 0.4 SSR07782 4 0.4 NN0228009 4 0.4 NN0224680 4 0.7 NN02269734 2.4 SSR01949 4 4.1 SSR07209 4 4.1 NN0247583 4 4.1 NN0228802 4 4.1SSR19115 4 4.4 NN0246810 4 4.4 NN0228736 4 4.4 NN0247706 4 5.2 SSR144984 5.2 NN0225810 4 6.3 NN0224596 4 6.3 NN0225493 4 6.3 NN0247492 4 6.6SSR19380 4 9.6 SSR21062 4 9.6 NN0225975 4 9.6 SSR22231 4 9.6 NN0224796 49.8 NN0224711 4 9.8 NN0228893 4 9.8 SSR11074 4 12.5 SSR17427 4 12.5NN0223763 4 12.5 NN0225383 4 14.4 NN0228856 4 14.5 NN0228862 4 14.5NN0246870 4 20.0 NN0225260 4 20.3 SSR00012 4 20.6 NN0227442 4 20.6NN0223863 4 20.6 NN0225012 4 20.6 SSR17024 4 21.7 NN0226079 4 21.7NN0247573 4 21.7 NN0228250 4 21.9 NN0246309 4 22.1 NN0246393 4 24.6NN0229054 4 25.4 NN0225572 4 25.5 SSR13023 4 25.5 NN0228579 4 25.8SSR22706 4 25.8 NN0225143 4 25.8 NN0224450 4 26.8 NN0226440 4 27.9NN0226451 4 27.9 NN0223404 4 28.7 NN0226964 4 28.7 NN0225345 4 29.7NN0226204 4 29.8 NN0223589 4 29.8 NN0227018 4 29.9 NN0247182 4 30.1NN0227010 4 30.1 NN0247596 4 30.1 NN0226074 4 30.1 SSR02803 4 30.4SSR07236 4 30.4 NN0225088 4 30.4 NN0225150 4 30.4 NN0224867 4 30.5NN0224838 4 30.5 NN0224343 4 30.5 SSR21240 4 30.5 SSR33769 4 30.5NN0229028 4 30.5 NN0224864 4 30.5 NN0227999 4 31.3 NN0228029 4 31.3NN0227972 4 31.5 NN0224664 4 31.9 NN0223316 4 31.9 NN0224593 4 31.9NN0247603 4 31.9 SSR16892 4 33.0 NN0226218 4 33.0 NN0226219 4 33.0NN0247330 4 33.0 NN0224445 4 33.9 NN0227979 4 33.9 NN0224221 4 34.4NN0223413 4 35.3 NN0224932 4 35.5 NN0224414 4 35.5 NN0224320 4 35.5NN0228842 4 35.5 NN0228836 4 35.5 NN0247551 4 35.5 NN0227183 4 35.5NN0247666 4 35.6 NN0227879 4 35.8 NN0224535 4 35.8 NN0246369 4 35.8NN0223339 4 35.8 SSR01904 4 36.5 SSR16847 4 36.5 NN0223895 4 36.5NN0228796 4 37.3 SSR19044 4 37.3 NN0223620 4 37.5 NN0247100 4 37.6NN0247134 4 37.6 NN0246357 4 40.3 NN0246914 4 40.3 NN0225107 4 40.5SSR15731 4 42.1 SSR21644 4 42.1 NN0247712 4 42.1 NN0224464 4 42.6NN0228630 4 43.2 NN0224762 4 43.4 NN0224390 4 44.5 NN0229090 4 44.5NN0247725 4 45.8 NN0246832 4 46.0 NN0224211 4 46.0 NN0225000 4 46.5NN0225070 4 46.6 NN0225551 4 46.6 NN0247208 4 47.4 NN0225339 4 48.1SSR04385 4 49.5 NN0228715 4 49.5 NN0228035 4 49.8 NN0226692 4 49.9NN0224961 4 49.9 NN0226732 4 50.0 NN0227475 4 50.3 NN0225125 4 50.3NN0226656 4 50.5 NN0227120 4 50.5 NN0227873 4 50.5 NN0223888 4 50.5NN0229056 4 50.5 NN0247743 4 50.5 NN0228883 4 50.8 SSR11043 4 51.9NN0247689 4 51.9 NN0223084 4 53.7 SSR13456 4 53.7 NN0225084 4 53.9NN0226586 4 53.9 NN0247529 4 54.5 SSR17270 4 54.5 NN0247635 4 54.5NN0247342 4 54.5 NN0247771 4 54.5 NN0228536 4 54.8 NN0223375 4 56.2NN0226446 4 56.9 NN0224702 4 62.4 NN0224353 4 62.4 NN0227422 4 62.4NN0228031 4 62.4 NN0227154 4 62.4 NN0223414 4 62.4 NN0224265 4 65.5SSR05125 4 65.9 SSR07130 4 65.9 NN0224287 4 65.9 NN0226863 4 66.0NN0228562 4 66.0 NN0224234 4 66.0 NN0247077 4 66.0 NN0225482 4 66.5NN0225553 4 66.5 NN0224572 4 66.9 NN0224106 4 66.9 NN0224111 4 66.9NN0228930 4 66.9 NN0223654 4 66.9 NN0225081 4 67.1 NN0223940 4 67.1NN0227048 4 67.1 NN0246425 4 67.1 NN0224538 4 67.1 NN0228639 4 68.1SSR21501 4 69.1 NN0225543 4 69.1 NN0228032 4 69.1 NN0226556 4 69.1NN0226509 4 69.9 NN0227631 4 71.0 NN0227617 4 71.0 NN0223626 4 71.6NN0223094 4 71.6 NN0223832 4 71.6 NN0247543 4 72.0 NN0224652 4 74.4SSR07269 4 74.4 NN0224067 4 75.8 NN0226776 4 75.8 NN0227379 4 75.8NN0227315 4 75.8 NN0224041 4 75.8 SSR00299 4 75.8 NN0228602 4 75.8NN0223124 4 76.6 NN0223103 4 77.5 NN0225814 4 78.5 NN0228853 4 78.9NN0227015 4 78.9 NN0223942 4 79.9 NN0246738 4 81.7 NN0224495 4 82.0SSR01552 4 84.4 NN0225766 4 84.4 NN0247668 4 84.4 SSR22948 4 84.4NN0226086 4 84.4 SSR04534 4 84.4 NN0223539 4 84.6 NN0227261 4 84.6NN0227251 4 84.6 NN0226226 4 85.1 NN0246452 4 85.7 NN0226543 4 85.9NN0227762 4 85.9 NN0247486 4 85.9 SSR07550 4 86.2 NN0224069 4 86.2NN0227587 4 87.4 NN0227182 4 87.4 NN0227279 4 87.4 NN0227285 4 87.4NN0247726 4 87.6 SSR13159 4 87.6 NN0247782 4 87.6 NN0225419 4 88.0NN0227265 4 88.0 NN0223422 4 88.2 NN0246417 4 88.2 NN0247615 4 88.7NN0247555 4 88.7 NN0227211 4 88.7 SSR07543 4 89.4 NN0226481 4 89.4NN0226465 4 89.6 NN0224223 4 90.0 NN0226865 4 90.0 SSR17406 4 91.3NN0228648 4 91.3 SSR14257 4 91.3 NN0225826 4 91.6 NN0224777 4 92.0NN0224050 4 92.4 NN0227419 4 92.9 NN0228716 4 92.9 NN0223906 4 92.9NN0227718 4 94.3 SSR16315 4 94.3 NN0227942 4 94.5 NN0247421 4 94.5NN0225077 4 94.5 NN0223715 4 94.5 NN0223710 4 94.5 NN0228566 4 94.7NN0225541 4 94.7 NN0225476 4 94.7 NN0227586 4 95.7 NN0225015 4 96.5NN0228246 4 96.6 SSR02233 4 96.7 NN0225079 4 96.7 NN0226312 4 96.7NN0227403 4 96.7 SSR23770 4 96.7 NN0223754 4 97.3 NN0223896 4 97.5NN0226628 4 97.8 NN0225404 4 98.0 NN0225471 4 98.0 NN0224793 4 98.0NN0226220 4 98.0 NN0225734 4 98.1 NN0226011 4 98.2 NN0225208 4 98.2NN0228805 4 98.2 NN0223571 4 98.4 NN0224125 4 98.4 NN0224127 4 98.4NN0246631 4 98.4 SSR16292 4 98.9 SSR29080 4 98.9 NN0246416 4 98.9NN0246722 4 99.7 NN0246308 4 99.7 NN0247688 4 99.7 NN0247613 4 100.1SSR22155 4 100.2 NN0225987 4 100.2 NN0247623 4 100.4 NN0223759 4 100.5NN0227111 4 100.5 NN0223284 4 100.6 NN0223295 4 100.6 NN0224972 4 100.6NN0246321 4 100.6 NN0226554 4 100.6 SSR06347 4 100.6 NN0228925 4 100.6SSR15203 4 100.8 NN0226376 4 100.8 SSR21197 4 100.8 NN0226559 4 101.3SSR14015 4 101.3 NN0225554 4 101.4 NN0223718 4 101.4 NN0223742 4 101.4NN0227971 5 0.0 NN0226380 5 0.2 NN0224416 5 0.2 NN0224425 5 0.2NN0247032 5 0.4 NN0227159 5 0.4 NN0227081 5 0.5 SSR14247 5 0.7 NN02271635 0.7 SSR17022 5 0.9 NN0247383 5 0.9 NN0227849 5 1.0 NN0247369 5 1.0NN0227216 5 1.1 NN0227224 5 1.1 NN0226775 5 1.2 NN0226854 5 1.3 SSR130865 2.0 NN0227542 5 2.0 NN0224662 5 2.2 NN0223648 5 2.2 NN0223632 5 2.2NN0228445 5 2.3 NN0229042 5 2.3 NN0223496 5 2.5 NN0225635 5 2.5NN0224047 5 2.6 NN0223877 5 2.6 NN0224052 5 2.7 NN0227420 5 4.3NN0223296 5 5.1 SSR22469 5 5.1 SSR19538 5 5.9 NN0224657 5 5.9 SSR02454 59.6 NN0225945 5 9.6 NN0226455 5 9.6 SSR12467 5 9.6 SSR19719 5 9.6NN0226450 5 9.6 NN0226658 5 9.9 NN0224159 5 9.9 NN0226797 5 9.9NN0227362 5 9.9 NN0224785 5 9.9 NN0228078 5 9.9 NN0226536 5 9.9NN0226528 5 9.9 NN0228505 5 10.0 NN0226756 5 10.0 NN0223780 5 10.0NN0247311 5 10.2 NN0227594 5 10.2 SSR16032 5 10.2 NN0225849 5 10.2NN0227145 5 10.2 SSR10002 5 10.3 SSR16842 5 10.3 NN0223450 5 10.3NN0224763 5 14.2 NN0227468 5 14.6 NN0228918 5 14.6 NN0226418 5 14.6NN0225926 5 14.6 NN0225883 5 14.6 NN0228145 5 14.7 NN0227295 5 14.7NN0226387 5 14.7 NN0227584 5 15.0 NN0246349 5 15.1 NN0227168 5 15.8SSR01859 5 15.8 NN0227165 5 15.8 SSR00023 5 16.1 NN0226433 5 16.1SSR00398 5 16.1 NN0223950 5 16.4 SSR19998 5 16.4 NN0246612 5 17.6NN0224029 5 17.6 SSR01610 5 17.6 SSR20648 5 17.6 NN0246303 5 17.6SSR22172 5 17.6 NN0227653 5 17.6 NN0226303 5 17.6 NN0228175 5 17.7SSR07369 5 17.7 NN0227913 5 18.9 SSR20208 5 18.9 NN0246687 5 18.9NN0223949 5 18.9 NN0224861 5 18.9 NN0224841 5 18.9 NN0223182 5 19.0SSR10795 5 19.0 NN0225294 5 20.3 NN0225315 5 20.3 NN0229134 5 21.6NN0227380 5 21.6 NN0225199 5 21.7 NN0225904 5 21.7 NN0224426 5 21.8NN0226415 5 22.2 NN0225222 5 22.8 SSR13639 5 22.8 NN0225428 5 22.8NN0226841 5 22.8 SSR03341 5 24.5 SSR05819 5 24.5 SSR07559 5 24.5NN0228879 5 24.5 NN0226553 5 25.3 NN0223140 5 25.3 NN0227540 5 25.3NN0228457 5 25.6 NN0228069 5 26.2 SSR07057 5 26.7 NN0225531 5 26.7SSR10348 5 26.7 SSR03950 5 28.1 SSR11167 5 28.1 NN0228895 5 28.1NN0224856 5 29.0 NN0226092 5 29.9 NN0247223 5 30.1 NN0246356 5 30.4NN0224773 5 31.1 NN0225532 5 31.2 NN0225517 5 31.2 NN0229077 5 31.2SSR20895 5 31.2 NN0229137 5 31.2 NN0224304 5 31.7 NN0247509 5 31.7NN0227435 5 31.7 NN0227405 5 31.7 SSR23615 5 31.9 SSR10542 5 31.9SSR14051 5 31.9 SSR21861 5 31.9 NN0228317 5 31.9 NN0223802 5 32.3SSR20487 5 32.3 NN0227305 5 33.2 SSR18593 5 33.5 NN0223112 5 33.5NN0228982 5 33.5 NN0247183 5 33.5 NN0246332 5 33.8 NN0224476 5 33.9NN0224325 5 34.5 NN0227981 5 36.1 NN0247085 5 37.4 NN0226041 5 37.4NN0226671 5 37.4 NN0224375 5 37.6 NN0223399 5 38.6 SSR23101 5 39.1NN0224283 5 39.1 SSR11750 5 39.7 NN0224246 5 39.7 NN0247066 5 40.4NN0228739 5 40.4 NN0228814 5 40.5 NN0225803 5 40.9 NN0247244 5 41.0NN0223974 5 41.0 NN0224213 5 41.0 NN0224193 5 41.0 NN0224669 5 41.0SSR07284 5 41.0 NN0227571 5 41.2 NN0227555 5 41.2 NN0227525 5 41.3NN0223689 5 41.4 SSR14269 5 41.4 NN0223687 5 41.4 NN0226194 5 41.6NN0226187 5 41.6 NN0227343 5 42.3 NN0227129 5 42.3 NN0228943 5 42.3NN0228968 5 42.3 SSR19178 5 43.0 NN0225790 5 43.0 SSR10720 5 44.8NN0224377 5 44.8 SSR12921 5 44.8 NN0226709 5 44.8 NN0246677 5 45.2NN0224642 5 45.2 NN0247731 5 45.2 NN0224840 5 45.2 NN0226013 5 45.6NN0227895 5 46.1 NN0224349 5 46.1 SSR15893 5 47.8 NN0227665 5 47.8NN0226326 5 48.9 NN0229099 5 48.9 NN0225940 5 48.9 NN0225518 5 49.0NN0225544 5 49.1 NN0226346 5 49.2 NN0226508 5 49.2 NN0246770 5 49.3SSR07711 5 49.4 SSR21291 5 49.4 NN0223897 5 49.4 SSR23148 5 49.4SSR23132 5 49.4 SSR32717 5 49.4 NN0224455 5 49.4 SSR16110 5 49.4NN0226166 5 50.0 NN0228693 5 51.7 NN0246672 5 51.7 NN0226430 5 51.7NN0227495 5 52.3 NN0225480 5 53.2 NN0225654 5 53.2 NN0227497 5 53.2SSR15321 5 53.3 NN0223392 5 53.3 NN0224442 5 53.7 NN0225103 5 54.3NN0224698 5 54.3 NN0227581 5 55.5 NN0247308 5 55.6 NN0225661 5 55.9NN0226631 5 55.9 NN0224600 5 55.9 NN0246411 5 56.1 NN0223215 5 56.2NN0226316 5 56.2 NN0228725 5 56.3 NN0246410 5 56.3 NN0223921 5 56.5NN0229142 5 56.8 NN0226764 5 57.4 NN0224228 5 57.7 NN0224229 5 57.7NN0224406 5 58.3 NN0225311 5 58.6 NN0229148 5 59.9 Cs28 5 60.1 NN02277595 60.1 NN0223336 5 60.3 SSR15818 5 60.3 NN0225562 5 60.3 NN0247548 560.6 NN0224461 5 60.7 NN0227603 5 60.7 NN0246604 5 60.9 NN0247389 5 60.9NN0246347 5 61.4 NN0247348 5 61.9 NN0226809 5 62.6 NN0226762 5 62.6NN0247445 5 62.7 NN0223334 5 67.1 NN0225936 5 68.4 NN0225451 5 68.4NN0224327 5 68.4 NN0246449 5 68.4 NN0228465 5 68.4 NN0223507 5 68.6SSR02244 5 68.6 NN0246335 5 69.1 SSR06660 5 69.1 NN0224251 5 69.6SSR17464 5 69.6 NN0246571 5 69.6 NN0224832 5 71.1 SSR23706 5 71.3SSR03529 5 71.3 SSR07184 5 71.3 NN0226931 5 71.3 NN0228305 5 71.6NN0247786 5 71.8 SSR07100 5 71.8 NN0227082 5 71.8 SSR19172 5 71.8NN0247485 5 71.9 NN0227153 5 72.5 SSR00772 5 72.5 NN0227421 5 72.5NN0223866 5 72.5 SSR00670 5 73.6 SSR01498 5 73.6 SSR04816 5 73.6NN0224048 5 73.6 SSR20859 5 73.6 NN0227318 5 73.9 NN0226912 5 74.1NN0226871 5 74.4 NN0227924 5 74.4 NN0223640 5 74.4 NN0226104 5 74.4NN0223518 5 75.1 NN0247794 5 75.1 NN0226645 5 75.2 NN0223160 5 75.4NN0227504 5 75.5 NN0228997 5 75.5 NN0226525 5 75.5 NN0224248 5 75.5NN0224226 5 75.5 NN0228632 5 75.5 NN0247702 5 75.5 NN0229017 5 75.5NN0225600 5 75.5 NN0223755 5 75.6 NN0225793 5 75.7 NN0226053 5 75.7NN0246483 5 75.7 NN0246327 5 76.1 SSR14180 5 76.1 NN0226354 5 76.7NN0228464 5 76.7 SSR00648 5 76.9 CSWCT17 5 76.9 SSR26904 5 76.9NN0226186 5 76.9 NN0223789 5 77.0 NN0223809 5 77.0 NN0226623 5 77.2NN0223634 5 77.2 NN0246814 5 77.2 NN0227194 5 77.3 NN0225426 5 77.4NN0225876 5 77.4 NN0226156 5 77.4 NN0247406 5 77.6 NN0246437 5 77.8NN0223864 5 77.8 NN0246426 5 77.8 SSR17975 5 77.8 NN0223659 5 77.9NN0226257 5 77.9 NN0225344 5 77.9 SSR20486 5 77.9 NN0246586 5 77.9NN0226429 5 78.0 NN0225867 5 78.0 NN0225673 5 78.0 NN0228260 5 78.0NN0225717 5 78.0 NN0247058 5 78.1 NN0223685 5 78.1 NN0223699 5 78.1NN0223091 5 78.1 NN0226394 5 78.2 NN0225216 5 78.2 NN0247709 5 78.3NN0247443 5 78.3 NN0224090 5 78.3 NN0223081 5 78.3 SSR06303 5 78.6NN0225646 5 78.6 NN0225626 5 78.6 NN0229147 5 78.6 NN0229106 5 78.6NN0229020 5 78.6 NN0228957 5 78.6 NN0247140 5 78.6 NN0227071 5 78.7NN0246376 5 78.7 NN0224281 5 78.7 NN0226051 5 78.7 NN0226076 5 78.7NN0223859 5 79.1 NN0247574 5 79.1 SSR13237 5 79.3 NN0226937 5 79.3NN0226940 5 79.3 NN0247180 5 79.4 NN0247563 5 79.5 NN0247704 5 79.5NN0247447 5 79.5 NN0247630 5 79.5 NN0224507 5 80.0 NN0247388 5 80.1NN0224321 5 80.1 NN0225878 5 80.1 NN0224365 5 80.1 NN0225310 5 80.1NN0227929 5 80.3 NN0224031 5 80.6 NN0228710 5 80.7 NN0224991 5 80.7NN0226870 5 80.7 NN0228616 5 80.7 NN0226516 5 80.8 NN0226453 5 81.0NN0226540 5 81.4 NN0227287 5 81.5 NN0227228 5 81.5 SSR03514 5 81.5SSR21219 5 81.5 NN0228055 5 81.5 NN0246331 5 81.8 NN0227097 5 81.8NN0246889 5 81.8 NN0224713 5 81.8 NN0228255 5 81.8 NN0224443 5 81.8NN0228289 5 81.8 NN0225431 5 81.9 NN0227769 5 82.0 NN0226910 5 82.0NN0227655 5 82.0 SSR02895 5 82.1 SSR16143 5 82.1 NN0226391 5 82.1SSR19343 5 82.1 NN0247172 5 82.6 NN0228132 5 82.6 NN0226532 5 82.6NN0228148 5 82.7 SSR03758 5 82.7 NN0226082 5 82.7

Example 10 Development of QIR Mapping Population

A recombinant inbred line (RIL) mapping population, termed “QIR”,generated from an initial cross between the Downy Mildew susceptibleparent 05VA8409 (a line that was resistant to Powdery Mildew andCucurbit Yellow Stunting Disorder Virus, CYSDV) and a Downy Mildewresistant parent derived from PI-197088 was used to examine the geneticsof cucumber Downy Mildew (CDM) resistance. The 40 lines in thepopulation were phenotyped at F₄ and F₅ generations in Saint-Andiol,France. Previous QTL-mapping analyses with this data identified a majorQTL for CDM resistance on a linkage group, defined in this population as“IV,” which was later found to correspond to chromosome 5. Fine-mappingwith CAPS markers suggested two linked QTLs at 35.8 cM and 42.7 cM onthe genetic map generated from this population, within this region onchromosome 5.

Based on the three QTLs on chromosomes 2, 4, and 5, identified for CDMusing the API and VJ mapping populations (e.g. see Examples 7-8) whichshare the PI-197088 Downy Mildew resistant parent, the F₆ generation ofthis cross was genotyped with 60 markers spaced roughly evenly acrossthese three chromosomes, in order to validate these three QTLs in theQIR population. For each of the F₄ and F₅ generation trials, 10 matureplants of each Fn family were planted and tested for resistance tonatural infections of downy mildew (Pseudoperonospora cubensis) in arandomised block design of three replicates. For the F₄ trial, each F₄family received an overall disease score for each of the threereplicates. For the F₅ trial, each plant received a disease score foreach of the three replicates. Disease scores ranged from 2 (susceptible)to 8 (resistant). Sex determination was observed to be segregating inthe F₄ families. For analysis of the QIR mapping population, the rawphenotypic data from the F₄ and F₅ trials were adjusted to account forreplicate effects (and sex determination in the F₄). In all, 40 familieshad non-missing phenotype value in at least one of the F₄ or F₅ trials.

A total of 60 SNPs were genotyped on 301 F₆ samples corresponding to 38unique F₆ lines. Seven or eight plants of each RIL were genotyped, andboth phenotypic and genotypic data were used in this analysis. Eightsamples of the susceptible parent were also genotyped. Consensusgenotypes were deduced based on the following criterion: for each locus,if genotypes were not consistent across all replicate samples (plants ofthe same RIL), the genotype occurring in more than 50% of the sampleswas taken as the consensus; otherwise a “missing” genotype designationwas given to the locus. Of the 60 tested markers, 28 were monomorphic(not segregating) in the F₆ population and were excluded from furtheranalysis. Table 20 provides a summary of the markers used.

TABLE 20 Marker summary; 32 informative markers were genotyped across 38F6 RILs and used. Total # Max. Genotyped # Segregating Averageinter-marker inter-marker Chr Markers Loci spacing (cM) spacing (cM) 212 5 4.5 7.7 4 25 16 3.7 8.7 5 23 11 5.0 19.7 Total 60 32 4.3 19.7

A non-parametric approach based on the Kruskal-Wallis test (Kruskal andWallis, J. Amer. Statistical Assoc. 47: 583-621, 1952) and Haley-Knottregression (Haley & Knott, Heredity 69:315-324, 1992) was used to testfor the presence of a QTL at every 1 cM interval. Identical phenotypicvalues between RILs were assigned averaged ranks. Conditional genotypeprobabilities were estimated based on observed multiple-point markerdata using the Hidden Markov algorithm and the Kosambi map function.Significance was determined from permutation tests within 1,000permutations at α=0.05. All QTL-mapping analyses were performed usingR/QTL (Broman et al., Bioinformatics 19:889-890, 2003).

Example 11 QTL Analysis for QIR Population

Two different QTLs were identified at Chr5:55.5 cM and Chr4:70 cM forCDM resistance collected from the F₄ and F₅ QIR populations respectively(FIG. 6 & Table 21). No significant linkage to Downy Mildew resistancewas detected on Chr2 for either of the F₄ or F₅ datasets in the QIRpopulation. For both QTLs on chromosomes 4 and 5, resistance wasconferred by the allele corresponding to the resistant parent: eachresistant allele was estimated to reduce disease score by ˜0.7,suggesting an effect of 1.4 disease units per QTL (Table 21 & FIG. 7).

TABLE 21 QTLs for cucumber Downy Mildew resistance identified usingphenotypes from the F₄ and F₅ QIR populations. Generation F4 F5Chromosome 5 4 Position (cM) 55.5 69.8 LOD 3.65 2.58 P-value (1,000permutations) 0.000 0.026 % Variation Explained 36.80 25.53 AdditiveEffect −0.76 −0.70 Dominance Effect −1.25 1.09 Closest Marker NN0226631NN0246425 Distance to Closest Marker 0.0 2.7 Population Mean 3.34 3.88Donor Allele Mean 4.02 4.38 Susceptible Allele Mean 2.49 2.96 1-LODinterval (cM) 45.5-62.5 56.8-82.0 1-LOD Flanking MarkersNN0227981-NN0247786 NN0247342-NN0224495 2-LOD interval (cM) 41.5-77.531.8-82.0 2-LOD Flanking Markers NN0227981-NN0227071 NN0225088-NN02244953-LOD interval (cM) 28.5-78.3 25.8-82.0 3-LOD Flanking MarkersNN0224856-NN0227071 NN0228579-NN0224495

Because these two QTLs on chromosomes 4 and 5 have been consistentlyidentified in the API, VJ, and QIR populations, these two QTLs appear toact additively or epistatically in the genetic control of DownyMildew-resistance. The following epistatic model was tested: DownyMildew resistance ˜Q1+Q2+Q1×Q2, where Q1 corresponds to the Chr4:69.8 cMQTL and Q2 corresponds to the Chr5:55.5 cM QTL. While the full model wasfound to be significant for both the F₄ and F₅ data (P-value of theF-statistic was 0.016 and 0.015 respectively), examination of thesub-models revealed that only one term of the model was significant(P<0.05) in each case: only Q2 was significant for the F₄ dataset andonly Q1 was significant for the F₅ dataset.

Of the three QTLs (Chr2:21.8-73.8 cM, Chr4:13.8-87.6 cM, Chr5:22.6-81.0cM) previously identified using the API and VJ mapping populations, two(one on Chr4 and one on Chr5) were also identified in the current QIRpopulation. The two QTL regions are found to be relatively large. Thatis, even at 1-LOD intervals, the two QTLs are predicted to reside within˜20 cM. No significant epistatic interaction was identified betweenthese two QTLs, suggesting these two QTLs may have an additive effect onDowny Mildew resistance (FIG. 8).

Example 12 Fine Mapping of QTLs for Downy Mildew Resistance OnChromosomes 2, 4, and 5

Further fine mapping of QTLs of chromosomes 2, 4, and 5 conferringresistance to cucmber Downy Mildew was carried out in conjunction withthe QTL analyses described above (e.g. Table 19). Fine mapping with thefollowing markers on chromosome 2 (with map positions in parentheses):NNO246378 (14.554); NNO227646 (19.999); NNO223782 (22.448); NNO226279(26.487); NNO246831 (28.802); NNO226670 (30.115); NNO224124 (32.406);NNO247070 (34.669); NNO227137 (36.613); NNO247472 (38.441); NNO227700(55.622); and NNO247695 (66.466) indicated that the CDM QTL onchromosome 2 extends between map positions 14.5-66.4 cM, including14.5-38.4 cM, based on results from the QIR mapping population.

Fine mapping with the following markers on chromosome 4 (with mappositions in parentheses): NNO225012 (20.649); NNO228579 (25.816);NNO226451 (27.944); NNO225088 (30.428); NNO247551 (35.460); NNO247100(37.638); NNO224390 (44.531); NNO225551 (46.644); NNO228715 (49.546);NNO227475 (50.320); NNO227873 (50.477); NNO228883 (50.812); NNO223084(53.747); NNO226586 (53.869); NNO247342 (54.495); NNO224702 (62.385);NNO224265 (65.543); NNO225482 (66.478); NNO225553 (66.478); NNO223940(67.148); NNO246425 (67.148); NNO224041 (75.834); NNO224495 (81.973);NNO227587 (87.395); and NNO246631 (98.405) indicated that the CDM QTL onchromosome 4 extends between map positions 56.8-82.0 cM with a 1-LODinterval, or between 31.8-82.0 cM with a 2-LOD interval, or between25.8-82.0 cM with a 3-LOD interval, based on results from the QIRmapping population.

Fine mapping with the following markers on chromosome 5 (with mappositions in parentheses): NN5096749 (25.200); NNO228457 (25.235);NNO224856 (28.544); NNO246356 (30.032); NNO225532 (30.761); NNO228982(33.131); NNO227981 (35.718); NNO223689 (41.014); NNO225790 (42.626);NNO246677 (44.758); NNO247731 (44.780); NNO226326 (48.512); NN5096750(51.200); NN0246672 (51.326); NN0224442 (53.297); NN0226631 (55.452);NN0224600 (55.452); NN0246411 (55.649); NN0247786 (71.424); NN0223160(74.955); NN0223809 (76.617); NN0227071 (78.303); and NN0228148 (82.273)indicated that the CDM QTL on chromosome 5 extends between map positions45.5-62.5 cM with a 1-LOD interval, or between 41.5-77.5 cM with a 2-LODinterval, or between 28.5-78.3 cM with a 3-LOD interval, based onresults from the QIR mapping population.

Thus, the QTL of chromosome 2 may extend between map positions 14.5-66.4cM, or between 14.5-38.4 cM. Likewise, the QTL of chromosome 4 mayextend from 13.8-87.6 cM, or between 25.8-82.0 cM. or between 31.8-75.8cM. The QTL of chromosome 5 may likewise extend from 22.6-81.0 cM, orbetween 25.2-78.3 cM, or between 28.5-75.0 cM.

Example 13 Refinement of QTL Intervals for Downy Mildew Resistance andDevelopment of Backcross Lines with Genetic Recombination Events Acrossthe QTLs

In order to identify sub-region(s) from the previously identified QTLintervals (e.g. on chromosomes 2, 4, and/or 5) that are highlyassociated with DM resistance, introgression regions from PI197088 wereidentified in 61 DM resistant lines developed from two differentgermplasm classes. For both germplasm classes, the resistant linespresented introgressed regions in the three QTL regions, supporting theutility of the identified QTLs for conferring resistance to DM inbreeding material. For each QTL interval, regions with relatively highfrequency marker alleles found in PI197088 were observed; theseintrogressed regions are likely to contain the QTLs conferring DMresistance, or markers that are highly associated with these QTLs. Theregions highly associated with DM resistance were found to include:QTL2: 14.554-38.4 cM; QTL4:44.531-66.478 cM; and QTL5: 25.235-42.626 and53.297-71.424 cM.

A plant with resistance to DM, in this case, a progeny plant ofPI197088, referred to as the donor parent (DP), was crossed to plantsfrom several DM susceptible, elite inbred lines, referred to asrecurrent parents (“RP's”). Plants of the F₁ generation from such across were backcrossed to plants of each RP to form the backcross 1(BC₁) generation. In each RP pedigree, 40 to 50 BC₁ plants werebackcrossed to RP plants to form the BC₂ generation. This occurredbefore initial identification of the DM QTL.

When markers for the DM resistance QTL of chromosome 4 (“QTL04”) and theDM resistance QTL of chromosome 5 (“QTL05”) were developed, remnanttissue from each BC₁ plant was genotyped, and plants heterozygous forthe QTLs were identified. BC₂ progeny plants from DM QTL heterozygousBC₁ plants were genotyped with markers at the distal ends of each DMQTL. Using this data, BC₂ plants were selected that contained geneticrecombination events within one of the DM QTL, while displaying ahomozygous RP allele at the other DM QTL. These plants wereself-pollinated in a greenhouse to create the BC₂F₂ generation. In thefollowing crop cycle, BC₂F₂ plants from each recombinant BC₂ pedigreewere genotyped with markers in the DM QTL, selected for homozygosity ofthe favorable allele in the recombinant section of the DM QTL, and selfpollinated to create the BC₂F₃ generation. Each BC₂F₃ family wasuniformly homozygous for the DP allele in the recombinant section of oneDM QTL, and homozygous RP for the other QTL, in order to confer auniform phenotype for DM within each family. The recombinant BC₂F₃families could then be phenotyped for DM with four experimental controlsconsisting of BC₂F₃ lines that had been selected for presence of onlyunfavorable (RP) alleles at both QTL04 and QTL05 (null control);presence of unfavorable allele at QTL04, favorable allele at QTL05;presence of favorable allele at QTL04, unfavorable allele at QTL05;presence of favorable allele at both QTL04 and QTL05. Data can then beanalyzed in a series of paired tests between the controls and the BC₂F₃families. The genotypes of BC₂F₃ recombinant families showing DMresistance significantly better than the null control are evaluated forlocation of the region containing the favorable allele in the DM QTL. Ifall BC₂F₃ families sharing this genotype show superior DM resistance,that genetic interval is defined as containing the DM QTL. Throughcomparison of multiple BC₂F₃ families with different, but overlappingrecombinant fragments, a smaller genetic and physical region containingthe DM QTL is identifed. Additional genetic markers utilized in refiningthe map positions of the DM QTLs are listed in Table 22. Such markersare utilized with various mapping populations, such as QIR, API, or VJ,among others.

TABLE 22 Exemplary additional C. sativus genomic DNA sequencesflanking the sites of SNP markers linked to QTLs onchromosomes 2, 4, and 5, for refining QTL map positions. Chromosome;Marker map Genomic DNA Sequence (SEQ ID NOs: 70-87). SNP  Name positionsite with polymorphism is given within brackets. NN0246378 2; 14.554AATGACTTATCTAGATGGATATGATAACTTCACCGATTGTTGGATAAAAAAGCAGGTGAC[A/G]AGTTGTGTTATTATTGGGCAGTGATGTATTGTTAGnTAACCACTGAAATTGTTAAAATTA NN0224041 4: 75.834TCTTTAAAAGAAGCTTGAAAGATGAAGAAATTGCAAAATTTCAATCATTTCGATCCCTCC[A/T]CTCACCTGAAAAAGTGGTTAATTCTGATGATTTTAGATCATGGTCCATTGATCCCTCAAT NN0227587 4; 87.395ATTTTTAGATGTGAGCCTATTTATGTGCTCTTAACTTCTCTTTTAGTAATGGTGGAAATG[T/C]AATTTGTTTATGAGCAATGGTATCGTGAATAAATGGTTGGATACATAATAGCTTTGCTTG NN5096749 5; 25.200ACTAGGTCGTGGGATGTCGTTGGGGGACATGTTAAATCCATGGCTGTCTA[G/A]AAGGAATTTCCATATCATTGAGATCTAGGTGCACAAATTCC TCGAACCTA NN02248565; 28.544 GGACATTCTTCACATAAATCAGAATTGTCGTACACAACCCAAAATTCTCCAATTAGCTAA[T/C]AGCGTTACAGATCTTCTTTTCCGCTTCTTTCCACGATGTATTGATATAGTGTGCCCTGAA NN0223160 5; 74.955AATGCAACAATTCTTTCAGCTAAGGAGAAGAAAGAAGCAAAAGAAAGAAAACACTCCACC[T/C]CTAGCCATTGCCCATCATCCATCTAAATTTCTTACTAAGATGCATAATATCTTCCACATA NN0223809 5; 76.617GGAGATTGTTGCACGCCTGAAGAAAGCCTTCAGAATTACCATTAAGACTTCCCAACTCTG[T/C]CTGCATATTGTCAAGAAAGCTAGAGATTTTCTCAGAAGCTACTTTAATGGCAGGGCCCTC NN0226631 5; 55.452GTCAAAAAGGTCAAGTCCACACTCCTCAGCTTATGAATCAAAACCCTGCACAGAAGGGAA[A/T]CACAACAACATTTTCAACTTTTCAGCTTAACTTTTGGCAATCATCAGGTTAAACAGACTT NN0227981 5; 35.718GGATTAATTTGCAAAAACCTTAAGTCGGGGAATTAGGGCTAAAGAGGAATTAAGGTAGAA[T/G]TGATTACCCCTGAGGATTATTGAAGGATAAATTGAGTTGTGATTGATTAGCGAAATAACC NN0247786 5; 71.424TTAGGTTCCCGCTATTGCAATACCAACAAGGCATGAAGGCTTT[C/G]GCCACAATGTGAAATCATCAACAGTTACTATGAACAATGAATTCGCTA ACAGGAAAnCGT NN02464254; 67.148 GAAGTAAGTTGCCTGCTTCTCTTTTTTTCACTAGGAATTGCTATTCAGACAGTTATATGC[A/T]CATTTCCAATGGTGCTTTTTTGTTCATTGTTTTCAATGTTGGGATTGACATCTGTCTGAA NN0224495 4; 81.973ACGAATTTTCTAGTTGAGTCAAGTCAGGTTGGTTGGAAAGTGTTATCAGAATATGTCAGT[A/G]CTTGTCAACTCTCGCACTCTCTTTTGAGGCTATAAAGTTAGAGAAGAGTCTCTAGGAAGG NN0225088 4; 30.428GAGTTTCCAAAATTGAACATCTTCAATGGACAGAAAGTTTTGAAGTAGCAAGACTTAAGG[T/C]TTCCACTTGGTGTTCCTTATCTAAGTTCTCGAACAATTTGCATTTGATTTCTTAAATATT NN0227071 5; 78.303AGGAAAATACCTTCCAATAGAAAACATGGTAAATGCAATAAGCATCTAGTTCATCCAATT[C/G]CAAGGGTAATGGCTAGTTCAAGATGGAACTAAAACTCTCAGAGTGGTGTGTGCAATCCTG NN0223782 2; 22.448TTCATACGCCGTTGCAGCTGAAAGTGGCAACCATTACTTCCAGATCATTATGACAATAAA[T/C]AACCAAGGCCACCTTCATGCATAAATGGTAAGATAATAGCAAGCTCTATACCTTCTTTTT NN0228579 4; 25.816ACTCATATTTACAGAAAACTTACTCTAAACCACAAGTCCTTAACAAATATATTTCTGCTT[C/G]CGGCTCTCTTCCTATCATGAAATTTTGCAAG CTATTCAGAAAATCCNN0247342 4; 54.495 GTACTTGTACCAATATGAAATGTGCACATGCGCTCTTGTCCTAGATAATATGCACAGTTC[A/T]CCTTAAAAGCTAAGCATAAGCCACAAATCCAAGAACCAATGTAACCAAAACAGTATGGGA NN0247731 5; 44.780TGAAGAAGAGCCTTTATTGCGTCATCTGGAACCTCCTTTATCCATTTATCTTGAATTGGT[T/C]GGTCATGAACACACCTTTACCATTTATTATTCAATGCCATGATTGTCTTACACAGGTGTG

Initial mapping of the DM QTL used data from two F₅ populations referredto as the API and VJ populations. Mean DM scores from several trials forfamilies in the API population were tested for significant differencesfrom the population mean. Families with DM scores significantly higheror lower than the population mean were selected. Plants from each ofthese families were genotyped and selected for homozygosity across theDM QTL. The selected plants would also be genotyped for genome-widemarkers to define, in particular, regions that shifted from heterozygousin the F₅ family to homozygous in the selected F₅ plant and derived F₆family. These plants were self pollinated to create F₆ families. Thesefamilies are phenotyped along with experimental controls of theDM-susceptible parent of the API population, the DM-resistant parent ofthe population (PI197088), and the four experimental controls describedpreviously, consisting of BC₂F₃ lines that had been selected forpresence of only unfavorable (RP) alleles at both QTL04 and QTL05 (nullcontrol); presence of unfavorable allele at QTL04, favorable allele atQTL05; presence of favorable allele at QTL04, unfavorable allele atQTL05; presence of favorable allele at both QTL04 and QTL05. Thisanalysis allows for improved resolution of the genetic positions of theQTL intervals, and detection of additional QTL contributing to DMresistance. By changing genotypes in DM QTL from heterozygous in the F₅family to homozygous in the derived F₆ more uniform DM phenotypes aredisplayed, thus enhancing resolution of genotype-phenotype associations.

1. A method of obtaining cucumber germplasm comprising the steps of: a)assaying cucumber plants for the presence of at least a first geneticmarker genetically linked to a QTL that confers resistance to DownyMildew; and b) selecting at least a first cucumber plant comprising thegenetic marker and the QTL that confers resistance to Downy Mildew;wherein the QTL maps to a position between the sequence represented bySNP marker NN0228457 and SNP marker NN0228148, which map toapproximately 25.2 cM and 82.7 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0225012 and SNP markerNN0227587 which map to approximately 20.6 cM and 87.4 cM on the geneticmap of the linkage group termed cucumber chromosome 4; or wherein theQTL maps to a position between the sequence represented by SNP markerNN0223782 and SNP marker NN0226638 which map to approximately 22.5 cMand 88.4 cM on the genetic map of the linkage group termed cucumberchromosome
 2. 2. The method of claim 1, wherein the QTL maps to aposition between the sequence represented by SNP marker NN0227981 andSNP marker NN0226631, which map to approximately 35.7 cM and 55.5 cM onthe genetic map of the linkage group termed cucumber chromosome 5; orwherein the QTL maps to a position between the sequence represented bySNP marker NN0227981 and SNP marker NN0247786, which map toapproximately 35.7 cM and 71.4 cM on the genetic map of the linkagegroup termed cucumber chromosome
 5. 3. The method of claim 1, whereinthe QTL maps to a position between the sequence represented by SNPmarker NN0228579 and SNP marker NN0224495 which map to approximately25.8 cM and 82.0 cM on the genetic map of the linkage group termedcucumber chromosome 4; wherein the QTL maps to a position between thesequence represented by SNP marker NN0225088 and SNP marker NN0224041which map to approximately 30.4 cM and 75.8 cM on the genetic map of thelinkage group termed cucumber chromosome 4; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0228579 andSNP marker NN0224495 which map to approximately 54.5 cM and 82.0 cM onthe genetic map of the linkage group termed cucumber chromosome
 4. 4.The method of claim 1, wherein the QTL maps to a position between thesequence represented by SNP marker NN0246378 and SNP marker NN0246472which map to approximately 14.6 cM and 38.4 cM on the genetic map of thelinkage group termed cucumber chromosome
 2. 5. The method of claim 1,wherein the allele which confers resistance to Downy Mildew is derivedfrom cucumber line PI197088, or a progeny plant thereof.
 6. The methodof claim 1, wherein selecting the first cucumber plant further comprisesselecting the plant based on the presence of a plurality of geneticmarkers that map to a position between the sequence represented by SNPmarker NN0228457 and SNP marker NN0228148, which map to approximately25.2 cM and 82.7 cM on the genetic map of the linkage group termedcucumber chromosome 5; or wherein the QTL maps to a position between thesequence represented by SNP marker NN0227981 and SNP marker NN0226631,which map to approximately 35.7 cM and 55.5 cM on the genetic map of thelinkage group termed cucumber chromosome 5; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0227981 andSNP marker NN0247786, which map to approximately 35.7 cM and 71.4 cM onthe genetic map of the linkage group termed cucumber chromosome 5; orwherein the QTL maps to a position between the sequence represented bySNP marker NN0225012 and SNP marker NN0227587 which map to approximately20.6 cM and 87.4 cM on the genetic map of the linkage group termedcucumber chromosome 4; or wherein the QTL maps to a position between thesequence represented by SNP marker NN0247342 and SNP marker NN0224495which map to approximately 54.5 cM and 82.0 cM on the genetic map of thelinkage group termed cucumber chromosome 4; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0223782 andSNP marker NN0226638 which map to approximately 22.5 cM and 88.4 cM onthe genetic map of the linkage group termed cucumber chromosome
 2. 7.The method of claim 1, wherein the genetic marker is selected from thegroup consisting of markers NN0223782, NN0225385, NN0226670, NN0224124,NN0246472, NN0225358, NN0227700, NN0224617, NN0247695, NN0227242,NN0223824, NN0223181, NN0226638, NN0225012, NN0228579, NN0226451,NN0225088, NN0226219, NN0247551, NN0246357, NN0225551, NN0226732,NN0247689, NN0247342, NN0224702, NN0225482, NN0224538, NN0247543,NN0224041, NN0228853, NN0227762, NN0227587, NN0228457, NN0246356,NN0246332, NN0223399, NN0223689, NN0247731, NN0226166, NN0225480,NN0246411, NN0227759, NN0247348, NN0228465, NN0247786, NN0226645,NN0223809, NN0227071, NN0226870, NN0228148, NN0246378, NN0224041,NN0227587, NN5096749, NN0224856, NN0223160, NN0223809, NN0226631,NN0227981, NN0247786, NN0246425, NN0224495, NN0225088, NN0227071,NN0223782, NN0228579, NN0247342, and NN0247731, comprising a singlenucleotide polymorphism of one of SEQ ID NOs:20-87.
 8. The method ofclaim 7, wherein the genetic marker is selected from the groupconsisting of markers NN0223782, NN0225385, NN0226670, NN0224124,NN0246472, NN0225358, NN0227700, NN0224617, NN0247695, NN0227242,NN0223824, NN0223181, NN0226638, and NN0246378.
 9. The method of claim7, wherein the genetic marker is selected from the group consisting ofmarkers NN0225012, NN0228579, NN0226451, NN0225088, NN0226219,NN0247551, NN0246357, NN0225551, NN0226732, NN0247689, NN0247342,NN0224702, NN0225482, NN0224538, NN0247543, NN0224041, NN0228853,NN0227762, NN0227587, NN0224041, NN0227587, NN0246425, NN0224495,NN0225088, NN0228579, and NN0247342.
 10. The method of claim 7, whereinthe genetic marker is selected from the group consisting of markersNN0228457, NN0246356, NN0246332, NN0223399, NN0223689, NN0247731,NN0226166, NN0225480, NN0246411, NN0227759, NN0247348, NN0228465,NN0247786, NN0226645, NN0223809, NN0227071, NN0226870, NN0228148,NN5096749, NN0224856, NN0223160, NN0223809, NN0226631, NN0227981,NN0247786, NN0227071, and NN0247731.
 11. The method of claim 1, whereinassaying the cucumber plants comprises PCR, single strand conformationalpolymorphism analysis, denaturing gradient gel electrophoresis, cleavagefragment length polymorphism analysis, TAQMAN assay, and/or DNAsequencing.
 12. The method of claim 1, wherein the genetic marker mapswithin 20 cM, 10 cM or 1 cM of a QTL which confers resistance to DownyMildew
 13. The method of claim 1, wherein the genetic marker isNN0226631 or NN0246425.
 14. A method of cucumber plant breedingcomprising: a) assaying cucumber plants for the presence of at least afirst genetic marker genetically linked to a QTL that confers resistanceto Downy Mildew; and b) selecting at least a first cucumber plantcomprising the genetic marker and the QTL that confers resistance toDowny Mildew; wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0228457 and SNP marker NN0228148, which mapto approximately 25.2 cM and 82.7 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0227981 and SNP markerNN0226631, which map to approximately 35.7 cM and 55.5 cM on the geneticmap of the linkage group termed cucumber chromosome 5; wherein the QTLmaps to a position between the sequence represented by SNP markerNN0227981 and SNP marker NN0247786, which map to approximately 35.7 cMand 71.4 cM on the genetic map of the linkage group termed cucumberchromosome 5; wherein the QTL maps to a position between the sequencerepresented by SNP marker NN0225012 and SNP marker NN0227587 which mapto approximately 20.6 cM and 87.4 cM on the genetic map of the linkagegroup termed cucumber chromosome 4; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0247342 and SNP markerNN0224495 which map to approximately 54.5 cM and 82.0 cM on the geneticmap of the linkage group termed cucumber chromosome 4; or wherein theQTL maps to a position between the sequence represented by SNP markerNN0223782 and SNP marker NN0226638 which map to approximately 22.5 cMand 88.4 cM on the genetic map of the linkage group termed cucumberchromosome 2; and c) crossing the first cucumber plant with itself or asecond cucumber plant to produce progeny cucumber plants comprising theQTL that confers resistance to Downy Mildew.
 15. The method of claim 14,wherein selecting at least a first cucumber plant further comprisesselecting the plant based on the presence of a plurality of geneticmarkers that map to a position between the sequence represented by SNPmarker NN0228457 and SNP marker NN0228148, which map to approximately25.2 cM and 82.7 cM on the genetic map of the linkage group termedcucumber chromosome 5; wherein the QTL maps to a position between thesequence represented by SNP marker NN0227981 and SNP marker NN0226631,which map to approximately 35.7 cM and 55.5 cM on the genetic map of thelinkage group termed cucumber chromosome 5; wherein the QTL maps to aposition between the sequence represented by SNP marker NN0227981 andSNP marker NN0247786, which map to approximately 35.7 cM and 71.4 cM onthe genetic map of the linkage group termed cucumber chromosome 5;wherein the QTL maps to a position between the sequence represented bySNP marker NN0225012 and SNP marker NN0227587 which map to approximately20.6 cM and 87.4 cM on the genetic map of the linkage group termedcucumber chromosome 4; wherein the QTL maps to a position between thesequence represented by SNP marker NN0247342 and SNP marker NN0224495which map to approximately 54.5 cM and 82.0 cM on the genetic map of thelinkage group termed cucumber chromosome 4; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0223782 andSNP marker NN0226638 which map to approximately 22.5 cM and 88.4 cM onthe genetic map of the linkage group termed cucumber chromosome
 2. 16.The method of claim 14, further comprising the step of d) selecting aprogeny plant comprising the allele which confers resistance to DownyMildew and crossing the progeny plant with itself or a third cucumberplant to produce additional progeny plants.
 17. The method of claim 16,wherein the method further comprises repeating step (d) about 2-10times.
 18. The method of claim 14, wherein the allele which confersresistance to Downy Mildew is derived from cucumber line PI197088, or aprogeny plant thereof
 19. The method of claim 14, wherein the geneticmarker is selected from the group consisting of markers NN0223782,NN0225385, NN0226670, NN0224124, NN0246472, NN0225358, NN0227700,NN0224617, NN0247695, NN0227242, NN0223824, NN0223181, NN0226638,NN0225012, NN0228579, NN0226451, NN0225088, NN0226219, NN0247551,NN0246357, NN0225551, NN0226732, NN0247689, NN0247342, NN0224702,NN0225482, NN0224538, NN0247543, NN0224041, NN0228853, NN0227762,NN0227587, NN0228457, NN0246356, NN0246332, NN0223399, NN0223689,NN0247731, NN0226166, NN0225480, NN0246411, NN0227759, NN0247348,NN0228465, NN0247786, NN0226645, NN0223809, NN0227071, NN0226870,NN0228148, NN0246378, NN0224041, NN0227587, NN5096749, NN0224856,NN0223160, NN0223809, NN0226631, NN0227981, NN0247786, NN0246425,NN0224495, NN0225088, NN0227071, NN0223782, NN0228579, NN0247342, andNN0247731, comprising a single nucleotide polymorphism of one of SEQ IDNOs:20-87.
 20. The method of claim 19, wherein the genetic marker isselected from the group consisting of markers NN0223782, NN0225385,NN0226670, NN0224124, NN0246472, NN0225358, NN0227700, NN0224617,NN0247695, NN0227242, NN0223824, NN0223181, NN0226638, and NN0246378.21. The method of claim 19, wherein the genetic marker is selected fromthe group consisting of markers NN0225012, NN0228579, NN0226451,NN0225088, NN0226219, NN0247551, NN0246357, NN0225551, NN0226732,NN0247689, NN0247342, NN0224702, NN0225482, NN0224538, NN0247543,NN0224041, NN0228853, NN0227762, NN0227587, NN0224041, NN0227587,NN0246425, NN0224495, NN0225088, NN0228579, and NN0247342.
 22. Themethod of claim 19, wherein the genetic marker is selected from thegroup consisting of markers NN0228457, NN0246356, NN0246332, NN0223399,NN0223689, NN0247731, NN0226166, NN0225480, NN0246411, NN0227759,NN0247348, NN0228465, NN0247786, NN0226645, NN0223809, NN0227071,NN0226870, NN0228148, NN5096749, NN0224856, NN0223160, NN0223809,NN0226631, NN0227981, NN0247786, NN0227071, and NN0247731.
 23. Themethod of claim 14, wherein assaying the cucumber plants comprises PCR,single strand conformational polymorphism analysis, denaturing gradientgel electrophoresis, cleavage fragment length polymorphism analysis,TAQMAN assay, and/or DNA sequencing.
 24. The method of claim 14, whereinthe genetic marker maps within 20 cM, 10 cM or 1 cM of a QTL whichconfers resistance to Downy Mildew.
 25. The method of claim 14, whereinthe genetic marker is NN0226631 or NN0246425.
 26. The method of claim14, wherein the cucumber plant comprising at least one allele whichconfers resistance to Downy Mildew demonstrates a reduction of foliarsymptoms of chlorotic and/or necrotic lesions of at least, or greaterthan, 25%, relative to a non-resistant control cucumber line.
 27. Anisolated nucleic acid probe or primer that hybridizes under conditionsof 5×SSC, 50% formamide, and 42° C. to a cucumber plant genomic regionmapping within 40 cM of a QTL which confers resistance to Downy Mildewand comprises a sequence which maps on cucumber chromosomes 2, 4, or 5,wherein the probe or primer comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs:20-87.
 28. A cucumber plantproduced by the method of claim 14, or a progeny plant thereof thatcomprises said genetic marker and QTL that confers resistance to DownyMildew.
 29. A part of the cucumber plant of claim 28, further defined aspollen, an ovule, a leaf, an embryo, a root, a root tip, an anther, aflower, a fruit, a stem, a shoot, a seed, a protoplast, a cell, and acallus.
 30. A seed that produced the plant of claim
 28. 31. A cucumberplant comprising at least a first introgressed cucumber chromosomalregion conferring resistance to Pseudoperonospora cubensis, wherein theregion is selected from the group consisting of: a Downy Mildewresistance contributing QTL region found on chromosome 2, a Downy Mildewresistance contributing QTL region found on chromosome 4, and a DownyMildew resistance contributing QTL region found on chromosome 5; furtherwherein the QTL maps to a position between the sequence represented bySNP marker NN0228457 and SNP marker NN0228148, which map toapproximately 25.2 cM and 82.7 cM on the genetic map of the linkagegroup termed cucumber chromosome 5; wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0225012 and SNP markerNN0227587 which map to approximately 20.6 cM and 87.4 cM on the geneticmap of the linkage group termed cucumber chromosome 4; or wherein theQTL maps to a position between the sequence represented by SNP markerNN0246378 and SNP marker NN0247695 which map to approximately 14.6 cMand 66.5 cM on the genetic map of the linkage group termed cucumberchromosome
 2. 32. The cucumber plant of claim 31, wherein the QTL mapsto a position between the sequence represented by SNP marker NN0227981and SNP marker NN0226631, which map to approximately 35.7 cM and 55.5 cMon the genetic map of the linkage group termed cucumber chromosome 5; orwherein the QTL maps to a position between the sequence represented bySNP marker NN0227981 and SNP marker NN0247786, which map toapproximately 35.7 cM and 71.4 cM on the genetic map of the linkagegroup termed cucumber chromosome
 5. 33. The cucumber plant of claim 31,wherein the QTL maps to a position between the sequence represented bySNP marker NN0228579 and SNP marker NN0224495 which map to approximately25.8 cM and 82.0 cM on the genetic map of the linkage group termedcucumber chromosome 4; wherein the QTL maps to a position between thesequence represented by SNP marker NN0225088 and SNP marker NN0224041which map to approximately 30.4 cM and 75.8 cM on the genetic map of thelinkage group termed cucumber chromosome 4; or wherein the QTL maps to aposition between the sequence represented by SNP marker NN0228579 andSNP marker NN0224495 which map to approximately 54.5 cM and 82.0 cM onthe genetic map of the linkage group termed cucumber chromosome
 4. 34.The cucumber plant of claim 31, wherein the QTL maps to a positionbetween the sequence represented by SNP marker NN0246378 and SNP markerNN0246472 which map to approximately 14.6 cM and 38.4 cM on the geneticmap of the linkage group termed cucumber chromosome
 2. 35. The cucumberplant of claim 31, comprising at least two introgressed cucumberchromosomal regions selected from said group.
 36. The cucumber plant ofclaim 31, wherein the first introgressed cucumber chromosomal regionconferring resistance to Pseudoperonospora cubensis comprises an allelepresent in PI197088.
 37. The cucumber plant of claim 36, comprising theQTL region found on chromosome 2, wherein the QTL is introgressed fromPI197088.
 38. The cucumber plant of claim 36 further defined ascomprising an allele from PI197088 at one or more of markers NN0246378,NN0223782, NN0246472, and NN0247695.
 39. The cucumber plant of claim 38,further defined as comprising at least a first allele not present inPI197088, wherein the allele is detected with the group of markerscomprising NN0247695, NN0246472, NN0223782, and NN0246378.
 40. Thecucumber plant of claim 36, comprising the Downy Mildew resistancecontributing QTL region found on chromosome 4, wherein the QTL isintrogressed from PI197088
 41. The cucumber plant of claim 40, furtherdefined as comprising: a) an allele from PI197088 at one or more ofmarkers selected from the group consisting of: NN0228579, NN0225088,NN0225551, NN0247342, NN0246425, NN0224041, NN0224495, and NN0227587; b)an allele which is not present in PI197088 of at least one markerselected from the group consisting of: NN0228579, NN0225088, NN0225551,NN0247342, NN0246425, NN0224041, NN0224495, and NN0227587; c) an allelefrom PI197088 at marker NN0246425, an allele not present in PI197088 atmarker NN0247342 and an allele not present in PI197088 at markerNN0224495; d) an allele from PI197088 at marker NN0246425, an allele notpresent in PI197088 at marker NN0225088 and an allele not present inPI197088 at marker NN0224495; or e) an allele from PI197088 at markerNN0246425, an allele not present in PI197088 at marker NN0228579, and anallele not present in PI197088 at marker NN0224495.
 42. The cucumberplant of claim 36, comprising the QTL region found on chromosome 5,wherein the QTL region is introgressed from PI197088.
 43. The cucumberplant of claim 42, further defined as comprising: a) an allele fromPI197088 at one or more of markers selected from the group consistingof: NN5096749, NN0224856, NN0227981, NN0247731, NN0226631, NN0247786,NN0223160, NN0223809, NN0227071, and NN0228148; b) an allele which isnot present in PI197088 of at least one marker selected from the groupconsisting of: NN0228148, NN0227071, NN0223809, NN0223160, NN0247786,NN0226631, NN0247731, NN0227981, NN0224856, and NN5096749; c) an allelefrom PI197088 at marker NN0226631, an allele not present in PI197088 atmarker NN0227981, and an allele not present in PI197088 at markerNN0247786; d) an allele from PI197088 at marker NN0226631, an allele notpresent in PI197088 at marker NN0227981, and an allele not present inPI197088 at marker NN0227071; or e) an allele from PI197088 at markerNN0226631, an allele not present in PI197088 at marker NN0224856, and anallele not present in PI197088 at marker NN0227071.
 44. The cucumberplant of claim 31, further defined as an agronomically elite plant. 45.The cucumber plant of claim 31, comprising two introgressed cucumberchromosomal regions conferring resistance to Pseudoperonospora cubensis,wherein the regions comprise a Downy Mildew resistance contributing QTLregion found on chromosome 4, and a Downy Mildew resistance contributingQTL region found on chromosome
 5. 46. The cucumber plant of claim 31,wherein the plant is homozygous for said chromosomal region.